Agents that engage antigen-presenting cells through dendritic cell asialoglycoprotein receptor (dc-asgpr)

ABSTRACT

The present invention includes compositions and methods for making and using anti DC-ASGPR antibodies that can, e.g., activate DCs and other cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/372,832, filed Apr. 2, 2019, which is a continuation of U.S. patentapplication Ser. No. 15/265,236, filed Sep. 14, 2016, now U.S. Pat. No.10,279,030, issued May 7, 2019, which is a continuation application ofU.S. patent application Ser. No. 14/254,206, filed Apr. 16, 2014, nowU.S. Pat. No. 9,453,074, issued Sep. 27, 2016, which is a continuationapplication of U.S. patent application Ser. No. 13/551,198, filed Jul.17, 2012, now U.S. Pat. No. 8,728,481, issued May 20, 2014, which is adivisional of U.S. patent application Ser. No. 12/025,010 filed Feb. 2,2008, now U.S. Pat. No. 8,236,934, issued Aug. 7, 2012, which claimspriority to U.S. Provisional Application Ser. No. 60/888,036, filed Feb.2, 2007, the contents of each of which are incorporated by referenceherein in their entirety.

STATEMENT OF FEDERALLY FUNDED RESEARCH

This invention was made with government support under Grant No. U19A1057234 awarded by the National Institutes of Health (NIH). Thegovernment has certain rights in the invention.

TECHNICAL FIELD OF THE INVENTION

The present invention relates in general to the field of agents thatengage antigen-presenting cells through dendritic cellasialoglycoprotein receptor (DC-ASGPR).

REFERENCE TO A SEQUENCE LISTING

The present application includes a Sequence Listing filed separately asrequired by 37 CFR 1.821-1.825.

BACKGROUND OF THE INVENTION

Without limiting the scope of the invention, its background is describedin connection with antigen presentation.

Dendritic Cells play a pivotal role in controlling the interface ofinnate and acquired immunity by providing soluble and intercellularsignals, followed by recognition of pathogens. These functions of DCsare largely dependent on the expression of specialized surfacereceptors, ‘pattern recognition receptors’ (PRRs), represented, mostnotably, by toll-like receptors (TLRs) and C-type lectins or lectin-likereceptors (LLRs) (1-3).

In the current paradigm, a major role of TLRs is to alert DCs to produceinterleukin 12 (IL-12) and other inflammatory cytokines for initiatingimmune responses. C-type LLRs operate as constituents of the powerfulantigen capture and uptake mechanism of macrophages and DCs (1).Compared to TLRs, however, LLRs might have broader ranges of biologicalfunctions that include cell migrations (4), intercellular interactions(5). These multiple functions of LLRs might be due to the facts thatLLRs, unlike TLRs, can recognize both self and non-self. However, thecomplexity of LLRs, including the redundancy of a number of LLRsexpressed in immune cells, has been one of the major obstacles tounderstand the detailed functions of individual LLRs. In addition,natural ligands for most of these receptors remain unidentified.Nonetheless, evidence from recent studies suggests that LLRs, incollaboration with TLRs, may contribute to the activation of immunecells during microbial infections (6-14).

Valladeau et al. (The Journal of Immunology, 2001, 167: 5767-5774)described a novel LLR receptor on immature human Dendritic Cells relatedto hepatic Asialoglycoprotein Receptor and demonstrated that itefficiently mediated endocytosis. DC-ASGPR mRNA was observedpredominantly in immune tissues—in DC and granulocytes, but not in T, B,or NK cells, or monocytes. DC-ASGPR species were restricted to theCD14-derived DC obtained from CD34-derived progenitors, while absentfrom the CD1a-derived subset. Both monocyte-derived DC and tonsillarinterstitial-type DC expressed DC-ASGPR protein, while Langerhans-typecells did not. Furthermore, DC-ASGPR was a feature of immaturity, asexpression was lost upon CD40 activation. In agreement with the presenceof tyrosine-based and dileucine motifs in the intracytoplasmic domain,mAb against DC-ASGPR was rapidly internalized by DC at 37° C. Finally,intracellular DC-ASGPR was localized to early endosomes, suggesting thatthe receptor recycles to the cell surface following internalization ofligand. These findings identified DC-ASGPR/human macrophage lectin as afeature of immature DC, and as another lectin important for thespecialized Ag-capture function of DC.

SUMMARY OF THE INVENTION

While DC-ASGPR is known to be capable of directing the internalizationof surrogate antigen into human DC, the invention uses novel biologicalactivities of DC-ASGPR to effect particularly desirable changes in theimmune system, some in the context of antigen uptake (e.g.,vaccination), others through the unique action of DC-ASGPR effectors(alone or in concert with other immune regulatory molecules) capable ofeliciting signaling through this receptor on DC, B cells, and monocytes.The invention disclosure reveals means of developing unique agentscapable of activating cells bearing DC-ASGPR, as well as the effect ofthe resulting changes in cells receiving these signals regards action onother cells in the immune system. These effects (either alone, or inconcert with other signals (i.e., co-stimulation)) are highly predictiveof therapeutic outcomes for certain disease states or for augmentingprotective outcomes in the context of vaccination.

The present invention includes compositions and methods for increasingthe effectiveness of antigen presentation by a DC-ASGPR-expressingantigen presenting cell by isolating and purifying a DC-ASGPR-specificantibody or fragment thereof to which a targeted agent is attached thatforms an antibody-antigen complex, wherein the agent is processed andpresented by, e.g., a dendritic cell, that has been contacted with theantibody-agent complex. In one embodiment, the antigen presenting cellis a dendritic cell and the DC-ASGPR-specific antibody or fragmentthereof is bound to one half of a Coherin/Dockerin pair. TheDC-ASGPR-specific antibody or fragment thereof may also be bound to onehalf of a Coherin/Dockerin pair and an antigen is bound to thecomplementary half of the Coherin/Dockerin pair to form a complex.Non-limiting examples agents include one or more peptides, proteins,lipids, carbohydrates, nucleic acids and combinations thereof.

The agent may one or more cytokine selected from interleukins,transforming growth factors (TGFs), fibroblast growth factors (FGFs),platelet derived growth factors (PDGFs), epidermal growth factors(EGFs), connective tissue activated peptides (CTAPs), osteogenicfactors, and biologically active analogs, fragments, and derivatives ofsuch growth factors, B/T-cell differentiation factors, B/T-cell growthfactors, mitogenic cytokines, chemotactic cytokines, colony stimulatingfactors, angiogenesis factors, IFN-α, IFN-β, IFN-γ, III, IL2, IL3, IL4,IL5, IL6, IL7, IL8, IL9, IL10, IL11, IL12, IL13, IL14, IL15, IL16, IL17,IL18, etc., leptin, myostatin, macrophage stimulating protein,platelet-derived growth factor, TNF-α, TNF-β, NGF, CD40L, CD137L/4-1BBL,human lymphotoxin-β, G-CSF, M-CSF, GM-CSF, PDGF, IL-1α, IL1-β, IP-10,PF4, GRO, 9E3, erythropoietin, endostatin, angiostatin, VEGF,transforming growth factor (TGF) supergene family include the betatransforming growth factors (for example TGF-β1, TGF-β2, TGF-β3); bonemorphogenetic proteins (for example, BMP-1, BMP-2, BMP-3, BMP-4, BMP-5,BMP-6, BMP-7, BMP-8, BMP-9); heparin-binding growth factors (fibroblastgrowth factor (FGF), epidermal growth factor (EGF), platelet-derivedgrowth factor (PDGF), insulin-like growth factor (IGF)); Inhibins (forexample, Inhibin A, Inhibin B); growth differentiating factors (forexample, GDF-1); and Activins (for example, Activin A, Activin B,Activin AB). In another embodiment, the agent comprises an antigen thatis a bacterial, viral, fungal, protozoan or cancer protein.

The present invention also includes compositions and methods forincreasing the effectiveness of antigen presentation by dendritic cellscomprising binding a DC-ASGPR-specific antibody or fragment thereof towhich an antigen is attached that forms an antibody-antigen complex,wherein the antigen is processed and presented by a dendritic cell thathas been contacted with the antibody-antigen complex. Another embodimentis the use of antibodies or other specific binding molecules directed toDC-ASGPR for delivering antigens to antigen-presenting cells for thepurpose of eliciting protective or therapeutic immune responses. The useof antigen-targeting reagents specific to DC-ASGPR for vaccination viathe skin; antigen-targeting reagents specific to DC-ASGPR in associationwith co-administered or linked adjuvant for vaccination or use forantigen-targeting (vaccination) purposes of specific antigens which canbe expressed as recombinant antigen-antibody fusion proteins.

Another embodiment includes a method for increasing the effectiveness ofdendritic cells by isolating patient dendritic cells; exposing thedendritic cells to activating amounts of anti-DC-ASGPR antibodies orfragments thereof and antigen to form antigen-loaded, activateddendritic cells; and reintroducing the antigen-loaded, activateddendritic cells into the patient. The antigen may be a bacterial, viral,fungal, protozoan or cancer protein. The present invention also includesan anti-DC-ASGPR immunoglobulin or portion thereof that is secreted frommammalian cells and an antigen bound to the immunoglobulin. Theimmunoglobulin is bound to one half of a cohesin/dockerin domain, or itmay also include a complementary half of the cohesin-dockerin bindingpair bound to an antigen that forms a complex with the modular rAbcarrier, or a complementary half of the cohesin-dockerin binding pairthat is a fusion protein with an antigen. The antigen specific domainmay be a full length antibody, an antibody variable region domain, anFab fragment, a Fab′ fragment, an F(ab)₂ fragment, and Fv fragment, andFabc fragment and/or a Fab fragment with portions of the Fc domain. Theanti-DC-ASGPR immunoglobulin may also be bound to a toxin selected fromwherein the toxin is selected from the group consisting of a radioactiveisotope, metal, enzyme, botulin, tetanus, ricin, cholera, diphtheria,aflatoxins, perfringens toxin, mycotoxins, shigatoxin, staphylococcalenterotoxin B, T2, seguitoxin, saxitoxin, abrin, cyanoginosin,alphatoxin, tetrodotoxin, aconotoxin, snake venom and spider venom. Theantigen may be a fusion protein with the immunoglobulin or boundchemically covalently or not.

The present invention also includes compositions and methods forincreasing the effectiveness of dendritic cells by isolating patientdendritic cells, exposing the dendritic cells to activating amounts ofanti-DC-ASGPR antibodies or fragments thereof and antigen to formantigen-loaded, activated dendritic cells; and reintroducing theantigen-loaded, activated dendritic cells into the patient. The agentsmay be used to engage DC-ASGPR, alone or with co-activating agents, toactivate antigen-presenting cells for therapeutic or protectiveapplications, to bind DC-ASGPR and/or activating agents linked toantigens, alone or with co-activating agents, for protective ortherapeutic vaccination. Another use of is the development of specificantibody V-region sequences capable of binding to and activatingDC-ASGPR, for use as anti-DC-ASGPR agents linked to toxic agents fortherapeutic purposes in the context of diseases known or suspected toresult from inappropriate activation of immune cells via DC-ASGPR and asa vaccine with a DC-ASGPR-specific antibody or fragment thereof to whichan antigen is attached that forms an antibody-antigen complex, whereinthe antigen is processed and presented by a dendritic cell that has beencontacted with the antibody-antigen complex.

BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the features and advantages of thepresent invention, reference is now made to the detailed description ofthe invention along with the accompanying figures and in which:

FIGS. 1A to 1E demonstrate signaling through lectin-like receptorDC-ASGPR activates DCs, resulting in increased levels of costimulatorymolecules as well as cytokines and chemokines. FIG. 1A shows three dayand six day GM/IL-4 DCs were stained with FITC-labeled goat anti-mouseIgG followed by mouse monoclonal anti-human DC-ASGPR, antibody. FIG. 1Bshows six day GM/IL-4 DCs were cultured in plates coated with theanti-DC-ASGPR or control mAbs (1-2 ug/ml) for 16-18 h. Cells werestained with anti-CD86 and HLA-DR antibodies labeled with fluorescentdyes. Open and filled bars in the histograms represent cells activatedwith isotype control mAbs and anti-lectin mAbs, respectively. FIG. 1Cshows six day GM/IL-4 DCs were cultured in plates coated with the mAbsfor 12 h, and subjected to RNA isolation and Affymetrix Gene Chipanalysis, as described in Methods. Fold increases of gene expression byanti-lectin mAbs were compared with the gene expression levels in DCsstimulated with control mAbs. FIG. 1D shows the cytokines and chemokinesin the culture supernatants from the experiment shown in FIG. 1B weremeasured by Luminex. FIG. 1E shows six day GM/IL-4 DCs were cultured inplates coated with mAbs in the presence or absence of 50 ng/ml solubleCD40L, for 16-18 h, and then stained with anti-CD83 antibodies.Cytokines and chemokines in the culture supernatants from the experimentshown in FIG. 1E were measured by Luminex. Results shown arerepresentative of three independent experiments using cells fromdifferent normal donors.

FIGS. 2A to 2D shows that DC-ASGPR expressed on DCs, contributes toenhanced humoral immune responses. Six day GM/IL-4 DCs, 5×10³/well, wereincubated in 96 well plates coated with anti-DC-ASGPR or control mAb for16-18 h, and then 1×10⁵ autologous CD19⁺ B cells stained with CFSE wereco-cultured in the presence of 20 units/ml IL-2 and 50 nM CpG. FIG. 2Ais a FACS of day six cells stained with fluorescently labeledantibodies. CD3+ and 7-AAD⁺ cells were gated out. CD38⁺ and CFSE⁻ cellswere purified by FACS sorter and Giemsa staining was performed. FIG. 2Bare culture supernatants on day thirteen were analyzed for total IgM,IgG, and IgM by sandwich ELISA. FIG. 1C shows DCs pulsed with 5multiplicity of infection (moi) of heat-inactivated influenza virus(PR8), and cultured with B cells. Culture supernatant was analyzed forinfluenza-specific immunoglobulins (Igs) on day thirteen. FIG. 1D showsDC cultured with anti-DC-ASGPR or control mAb were stained for cellsurface APRIL expression and the supernatants assayed for soluble APRIL.

FIGS. 3A to 3D shows the cell surface expression of DC-ASGPR on B cellscontribute to B cell activation and immunoglobulin production. FIG. 3Aare PBMCs from buffy coats were stained with anti-CD19, anti-CD3, andanti-DC-ASGPR or control mAb. CD19⁺ and CD3⁺ cells were gated and theexpression levels of the molecules on CD19⁺ B cells were measured byflow cytometry. FIG. 3B are CD19⁺ B cells from buffy coats were culturedin plates coated with the mAbs for 12 h, and subjected to RNA isolationand Affymetrix Gene Chip analysis as described in Methods. Foldincreases of gene expression by anti-DC-ASGPR mAb were compared to thegene expression levels in CD19⁺ B cells stimulated with control mAb.FIG. 3C shows CD19⁺ B cells were cultured in plates coated with the mAbsfor 16-18 h, and then culture supernatants were analyzed for cytokinesand chemokines by Luminex. FIG. 3D shows 1×10⁵ CD19⁺ B cells werecultured in plates coated with the mAbs for thirteen days. Total Iglevels were measured by ELISA. Data are representative of two repeatexperiments using cells from three different normal donors.

FIGS. 4A to 4D shows that the proliferation of purified allogeneic Tcells was significantly enhanced by DCs stimulated with mAb specific forDC-ASGPR.

FIG. 5 shows that certain anti-DC-ASGPR mAbs can activate DC.GM-CSF/IL-4. DC were incubated for 24 hrs with one of a panel of 12 pureanti-ASGPR mAbs. Cells were then tested for expression of cell surfaceCD86 (a DC activation marker) and supernatants were assayed for secretedcytokines. Three mAbs (36, 38, 43) from the anti-ASGPR mAb panelactivated DC.

FIG. 6 shows that different antigens can be expressed in the context ofa DC-ASGPR rAb. Such an anti-DC-ASGPR rAb.Doc protein can be simplymixed with any Cohesin.fusion protein to assemble a stable non-covalent[rAb.Doc:Coh.fusion] complex that functions just as a rAb.fusionprotein.

FIG. 7—GM-CSF/IFNa DCs (5,000/well) were loaded with 10 or 1 nManti-DC-ASGPR.Doc:Coh.Flu M1, or hIgG4.Doc:Coh.Flu M1 complexes. After 6h, autologous CD8+ T cells (200,000/well) were added into the cultures.At day 8, the CD8+ T cells were analyzed for expansion of cells bearingTCR specific for a HLA-A201 immuno-dominant peptide. The inner boxesindicate the percentage of tetramer-specific CD8+ T cells.

FIGS. 8A-8D demonstrated the cross reactivity of the differentantibodies with monkey ASGPR.

DETAILED DESCRIPTION OF THE INVENTION

While the making and using of various embodiments of the presentinvention are discussed in detail below, it should be appreciated thatthe present invention provides many applicable inventive concepts thatcan be embodied in a wide variety of specific contexts. The specificembodiments discussed herein are merely illustrative of specific ways tomake and use the invention and do not delimit the scope of theinvention.

To facilitate the understanding of this invention, a number of terms aredefined below. Terms defined herein have meanings as commonly understoodby a person of ordinary skill in the areas relevant to the presentinvention. Terms such as “a”, “an” and “the” are not intended to referto only a singular entity, but include the general class of which aspecific example may be used for illustration. The terminology herein isused to describe specific embodiments of the invention, but their usagedoes not delimit the invention, except as outlined in the claims.

Dendritic cells (DCs) are antigen-presenting cells that play a key rolein regulating antigen-specific immunity (Mellman and Steinman 2001),(Banchereau, Briere et al. 2000), (Cella, Sallusto et al. 1997). DCscapture antigens, process them into peptides, and present these to Tcells. Therefore delivering antigens directly to DC is a focus area forimproving vaccines. One such example is the development of DC-basedvaccines using ex-vivo antigen-loading of autologous DCs that are thenre-administrated to patients (Banchereau, Schuler-Thurner et al. 2001),(Steinman and Dhodapkar 2001). Another strategy to improve vaccineefficacy is specific targeting to DC of antigen conjugated to antibodiesagainst internalizing DC-specific receptors. The potential of targetingDC for vaccination is highlighted by key mouse studies. In vivo,targeting with an anti-LOX-1 mAb coupled to ovalbumin (OVA) induced aprotective CD8+T cell response, via exogenous antigen cross-presentationtoward the MHC class I pathway (Delneste, Magistrelli et al. 2002).Also, OVA conjugated to anti-DEC205 mAb in combination with a CD40Lmaturation stimulus enhanced the MHC class I-restricted presentation byDCs in vivo and led to the durable formation of effector memory CD8+ Tcells (Bonifaz, Bonnyay et al. 2004). Both these studies showed dramaticdose-sparing (i.e., strong immune-responses at very low antigen doses)and suggested broader responses than normally seen with other types ofOVA immunization. Recent work with targeting of HIV gag antigen to DCvia DEC205 has extended these concepts to a clinically relevant antigenand confirmed the tenents of targeting antigen to DC—dramaticdose-sparing, protective responses from a single vaccination, andexpansion of antigen-specific T cells in both the CD8 and CD4compartments (Trumpfheller, Finke et al. 2006).

The present invention provides for the complexing of multiple antigensor proteins (engineered, expressed, and purified independently from theprimary mAb) in a controlled, multivariable fashion, to one singleprimary recombinant mAb. Presently, there are methods for engineeringsite-specific biotinylation sites that provide for the addition ofdifferent proteins (each engineered separately linked to streptavidin)to the one primary mAb. However, the present invention provides foraddition to the primary mAb of multiple combinations, in fixed equimolarratios and locations, of separately engineered proteins.

As used herein, the term “modular rAb carrier” is used to describe arecombinant antibody system that has been engineered to provide thecontrolled modular addition of diverse antigens, activating proteins, orother antibodies to a single recombinant monoclonal antibody (mAb). TherAb may be a monoclonal antibody made using standard hybridomatechniques, recombinant antibody display, humanized monoclonalantibodies and the like. The modular rAb carrier can be used to, e.g.,target (via one primary recombinant antibody against an internalizingreceptor, e.g., a human dendritic cell receptor) multiple antigensand/or antigens and an activating cytokine to dendritic cells (DC). Themodular rAb carrier may also be used to join two different recombinantmAbs end-to-end in a controlled and defined manner.

The antigen binding portion of the “modular rAb carrier” may be one ormore variable domains, one or more variable and the first constantdomain, an Fab fragment, a Fab′ fragment, an F(ab)₂ fragment, and Fvfragment, and Fabc fragment and/or a Fab fragment with portions of theFc domain to which the cognate modular binding portions are added to theamino acid sequence and/or bound. The antibody for use in the modularrAb carrier can be of any isotype or class, subclass or from any source(animal and/or recombinant).

In one non-limiting example, the modular rAb carrier is engineered tohave one or more modular cohesin-dockerin protein domains for makingspecific and defined protein complexes in the context of engineeredrecombinant mAbs. The mAb is a portion of a fusion protein that includesone or more modular cohesin-dockerin protein domains carboxy from theantigen binding domains of the mAb. The cohesin-dockerin protein domainsmay even be attached post-translationally, e.g., by using chemicalcross-linkers and/or disulfide bonding.

The term “antigen” as used herein refers to a molecule that can initiatea humoral and/or cellular immune response in a recipient of the antigen.Antigen may be used in two different contexts with the presentinvention: as a target for the antibody or other antigen recognitiondomain of the rAb or as the molecule that is carried to and/or into acell or target by the rAb as part of a dockerin/cohesin-moleculecomplement to the modular rAb carrier. The antigen is usually an agentthat causes a disease for which a vaccination would be advantageoustreatment. When the antigen is presented on MHC, the peptide is oftenabout 8 to about 25 amino acids. Antigens include any type of biologicmolecule, including, for example, simple intermediary metabolites,sugars, lipids and hormones as well as macromolecules such as complexcarbohydrates, phospholipids, nucleic acids and proteins. Commoncategories of antigens include, but are not limited to, viral antigens,bacterial antigens, fungal antigens, protozoal and other parasiticantigens, tumor antigens, antigens involved in autoimmune disease,allergy and graft rejection, and other miscellaneous antigens.

The modular rAb carrier is able to carry any number of active agents,e.g., antibiotics, anti-infective agents, antiviral agents, anti-tumoralagents, antipyretics, analgesics, anti-inflammatory agents, therapeuticagents for osteoporosis, enzymes, cytokines, anticoagulants,polysaccharides, collagen, cells, and combinations of two or more of theforegoing active agents. Examples of antibiotics for delivery using thepresent invention include, without limitation, tetracycline, aminoglycosides, penicillins, cephalosporins, sulfonamide drugs, chloramphenicolsodium succinate, erythromycin, vancomycin, lincomycin, clindamycin,nystatin, amphotericin B, amantidine, idoxuridine, p-amino salicyclicacid, isoniazid, rifampin, antinomycin D, mithramycin, daunomycin,adriamycin, bleomycin, vinblastine, vincristine, procarbazine, imidazolecarboxamide, and the like.

Examples of anti-tumor agents for delivery using the present inventioninclude, without limitation, doxorubicin, Daunorubicin, taxol,methotrexate, and the like. Examples of antipyretics and analgesicsinclude aspirin, Motrin®, Ibuprofen®, naprosyn, acetaminophen, and thelike.

Examples of anti-inflammatory agents for delivery using the presentinvention include, without limitation, include NSAIDS, aspirin,steroids, dexamethasone, hydrocortisone, prednisolone, Diclofenac Na,and the like.

Examples of therapeutic agents for treating osteoporosis and otherfactors acting on bone and skeleton include for delivery using thepresent invention include, without limitation, calcium, alendronate,bone GLa peptide, parathyroid hormone and its active fragments, histoneH4-related bone formation and proliferation peptide and mutations,derivatives and analogs thereof.

Examples of enzymes and enzyme cofactors for delivery using the presentinvention include, without limitation, pancrease, L-asparaginase,hyaluronidase, chymotrypsin, trypsin, tPA, streptokinase, urokinase,pancreatin, collagenase, trypsinogen, chymotrypsinogen, plasminogen,streptokinase, adenyl cyclase, superoxide dismutase (SOD), and the like.

Examples of cytokines for delivery using the present invention include,without limitation, interleukins, transforming growth factors (TGFs),fibroblast growth factors (FGFs), platelet derived growth factors(PDGFs), epidermal growth factors (EGFs), connective tissue activatedpeptides (CTAPs), osteogenic factors, and biologically active analogs,fragments, and derivatives of such growth factors. Cytokines may beB/T-cell differentiation factors, B/T-cell growth factors, mitogeniccytokines, chemotactic cytokines, colony stimulating factors,angiogenesis factors, IFN-α, IFN-β, IFN-γ, IL1, IL2, IL3, IL4, IL5, IL6,IL7, IL8, IL9, IL10, IL11, IL12, IL13, IL14, IL15, IL16, IL17, IL18,etc., leptin, myostatin, macrophage stimulating protein,platelet-derived growth factor, TNF-α, TNF-β, NGF, CD40L, CD137L/4-1BBL,human lymphotoxin-β, G-CSF, M-CSF, GM-CSF, PDGF, IL-1α, IL1-β, IP-10,PF4, GRO, 9E3, erythropoietin, endostatin, angiostatin, VEGF or anyfragments or combinations thereof. Other cytokines include members ofthe transforming growth factor (TGF) supergene family include the betatransforming growth factors (for example TGF-β1, TGF-β2, TGF-β3); bonemorphogenetic proteins (for example, BMP-1, BMP-2, BMP-3, BMP-4, BMP-5,BMP-6, BMP-7, BMP-8, BMP-9); heparin-binding growth factors (forexample, fibroblast growth factor (FGF), epidermal growth factor (EGF),platelet-derived growth factor (PDGF), insulin-like growth factor(IGF)); Inhibins (for example, Inhibin A, Inhibin B); growthdifferentiating factors (for example, GDF-1); and Activins (for example,Activin A, Activin B, Activin AB).

Examples of growth factors for delivery using the present inventioninclude, without limitation, growth factors that can be isolated fromnative or natural sources, such as from mammalian cells, or can beprepared synthetically, such as by recombinant DNA techniques or byvarious chemical processes. In addition, analogs, fragments, orderivatives of these factors can be used, provided that they exhibit atleast some of the biological activity of the native molecule. Forexample, analogs can be prepared by expression of genes altered bysite-specific mutagenesis or other genetic engineering techniques.

Examples of anticoagulants for delivery using the present inventioninclude, without limitation, include warfarin, heparin, Hirudin, and thelike. Examples of factors acting on the immune system include fordelivery using the present invention include, without limitation,factors which control inflammation and malignant neoplasms and factorswhich attack infective microorganisms, such as chemotactic peptides andbradykinins.

Examples of viral antigens include, but are not limited to, e.g.,retroviral antigens such as retroviral antigens from the humanimmunodeficiency virus (HIV) antigens such as gene products of the gag,pol, and env genes, the Nef protein, reverse transcriptase, and otherHIV components; hepatitis viral antigens such as the S, M, and Lproteins of hepatitis B virus, the pre-S antigen of hepatitis B virus,and other hepatitis, e.g., hepatitis A, B, and C, viral components suchas hepatitis C viral RNA; influenza viral antigens such as hemagglutininand neuraminidase and other influenza viral components; measles viralantigens such as the measles virus fusion protein and other measlesvirus components; rubella viral antigens such as proteins E1 and E2 andother rubella virus components; rotaviral antigens such as VP7sc andother rotaviral components; cytomegaloviral antigens such as envelopeglycoprotein B and other cytomegaloviral antigen components; respiratorysyncytial viral antigens such as the RSV fusion protein, the M2 proteinand other respiratory syncytial viral antigen components; herpes simplexviral antigens such as immediate early proteins, glycoprotein D, andother herpes simplex viral antigen components; varicella zoster viralantigens such as gpI, gpII, and other varicella zoster viral antigencomponents; Japanese encephalitis viral antigens such as proteins E,M-E, M-E-NS1, NS1, NS1-NS2A, 80% E, and other Japanese encephalitisviral antigen components; rabies viral antigens such as rabiesglycoprotein, rabies nucleoprotein and other rabies viral antigencomponents. See Fundamental Virology, Second Edition, eds. Fields, B. N.and Knipe, D. M. (Raven Press, New York, 1991) for additional examplesof viral antigens.

Antigenic targets that may be delivered using the rAb-DC/DC-antigenvaccines of the present invention include genes encoding antigens suchas viral antigens, bacterial antigens, fungal antigens or parasiticantigens. Viruses include picornavirus, coronavirus, togavirus,flavirvirus, rhabdovirus, paramyxovirus, orthomyxovirus, bunyavirus,arenavirus, reovirus, retrovirus, papilomavirus, parvovirus,herpesvirus, poxvirus, hepadnavirus, and spongiform virus. Other viraltargets include influenza, herpes simplex virus 1 and 2, measles,dengue, smallpox, polio or HIV. Pathogens include trypanosomes,tapeworms, roundworms, helminthes, and malaria. Tumor markers, such asfetal antigen or prostate specific antigen, may be targeted in thismanner. Other examples include: HIV env proteins and hepatitis B surfaceantigen. Administration of a vector according to the present inventionfor vaccination purposes would require that the vector-associatedantigens be sufficiently non-immunogenic to enable long term expressionof the transgene, for which a strong immune response would be desired.In some cases, vaccination of an individual may only be requiredinfrequently, such as yearly or biennially, and provide long termimmunologic protection against the infectious agent. Specific examplesof organisms, allergens and nucleic and amino sequences for use invectors and ultimately as antigens with the present invention may befound in U.S. Pat. No. 6,541,011, relevant portions incorporated hereinby reference, in particular, the tables that match organisms andspecific sequences that may be used with the present invention.

Bacterial antigens for use with the rAb vaccine disclosed hereininclude, but are not limited to, e.g., bacterial antigens such aspertussis toxin, filamentous hemagglutinin, pertactin, FIM2, FIM3,adenylate cyclase and other pertussis bacterial antigen components;diptheria bacterial antigens such as diptheria toxin or toxoid and otherdiptheria bacterial antigen components; tetanus bacterial antigens suchas tetanus toxin or toxoid and other tetanus bacterial antigencomponents; streptococcal bacterial antigens such as M proteins andother streptococcal bacterial antigen components; gram-negative bacillibacterial antigens such as lipopolysaccharides and other gram-negativebacterial antigen components, Mycobacterium tuberculosis bacterialantigens such as mycolic acid, heat shock protein 65 (HSP65), the 30 kDamajor secreted protein, antigen 85A and other mycobacterial antigencomponents; Helicobacter pylori bacterial antigen components;pneumococcal bacterial antigens such as pneumolysin, pneumococcalcapsular polysaccharides and other pneumococcal bacterial antigencomponents; haemophilus influenza bacterial antigens such as capsularpolysaccharides and other haemophilus influenza bacterial antigencomponents; anthrax bacterial antigens such as anthrax protectiveantigen and other anthrax bacterial antigen components; rickettsiaebacterial antigens such as rompA and other rickettsiae bacterial antigencomponent. Also included with the bacterial antigens described hereinare any other bacterial, mycobacterial, mycoplasmal, rickettsial, orchlamydial antigens. Partial or whole pathogens may also be: haemophilusinfluenza; Plasmodium falciparum; Neisseria meningitidis; Streptococcuspneumoniae; Neisseria gonorrhoeae; salmonella serotype typhi; shigella;Vibrio cholerae; Dengue Fever; Encephalitides; Japanese Encephalitis;Lyme disease; Yersinia pestis; west nile virus; yellow fever; tularemia;hepatitis (viral; bacterial); RSV (respiratory syncytial virus); HPIV 1and HPIV 3; adenovirus; small pox; allergies and cancers.

Fungal antigens for use with compositions and methods of the inventioninclude, but are not limited to, e.g., candida fungal antigencomponents; histoplasma fungal antigens such as heat shock protein 60(HSP60) and other histoplasma fungal antigen components; cryptococcalfungal antigens such as capsular polysaccharides and other cryptococcalfungal antigen components; coccidiodes fungal antigens such as spheruleantigens and other coccidiodes fungal antigen components; and tineafungal antigens such as trichophytin and other coccidiodes fungalantigen components.

Examples of protozoal and other parasitic antigens include, but are notlimited to, e.g., Plasmodium falciparum antigens such as merozoitesurface antigens, sporozoite surface antigens, circumsporozoiteantigens, gametocyte/gamete surface antigens, blood-stage antigen pf155/RESA and other plasmodial antigen components; toxoplasma antigenssuch as SAG-1, p30 and other toxoplasmal antigen components;schistosomae antigens such as glutathione-S-transferase, paramyosin, andother schistosomal antigen components; Leishmania major and otherleishmaniae antigens such as gp63, lipophosphoglycan and its associatedprotein and other leishmanial antigen components; and Trypanosoma cruziantigens such as the 75-77 kDa antigen, the 56 kDa antigen and othertrypanosomal antigen components.

Antigen that can be targeted using the rAb of the present invention willgenerally be selected based on a number of factors, including:likelihood of internalization, level of immune cell specificity, type ofimmune cell targeted, level of immune cell maturity and/or activationand the like. Examples of cell surface markers for dendritic cellsinclude, but are not limited to, MHC class I, MHC Class II, B7-2, CD18,CD29, CD31, CD43, CD44, CD45, CD54, CD58, CD83, CD86, CMRF-44, CMRF-56,DCIR and/or ASPGR and the like; while in some cases also having theabsence of CD2, CD3, CD4, CD8, CD14, CD15, CD16, CD19, CD20, CD56,and/or CD57. Examples of cell surface markers for antigen presentingcells include, but are not limited to, MHC class I, MHC Class II, CD40,CD45, B7-1, B7-2, IFN-γ receptor and IL-2 receptor, ICAM-1 and/or Fcγreceptor. Examples of cell surface markers for T cells include, but arenot limited to, CD3, CD4, CD8, CD 14, CD20, CD11b, CD16, CD45 andHLA-DR.

Target antigens on cell surfaces for delivery includes thosecharacteristic of tumor antigens typically will be derived from the cellsurface, cytoplasm, nucleus, organelles and the like of cells of tumortissue. Examples of tumor targets for the antibody portion of thepresent invention include, without limitation, hematological cancerssuch as leukemias and lymphomas, neurological tumors such asastrocytomas or glioblastomas, melanoma, breast cancer, lung cancer,head and neck cancer, gastrointestinal tumors such as gastric or coloncancer, liver cancer, pancreatic cancer, genitourinary tumors suchcervix, uterus, ovarian cancer, vaginal cancer, testicular cancer,prostate cancer or penile cancer, bone tumors, vascular tumors, orcancers of the lip, nasopharynx, pharynx and oral cavity, esophagus,rectum, gall bladder, biliary tree, larynx, lung and bronchus, bladder,kidney, brain and other parts of the nervous system, thyroid, Hodgkin'sdisease, non-Hodgkin's lymphoma, multiple myeloma and leukemia.

Examples of antigens that may be delivered alone or in combination toimmune cells for antigen presentation using the present inventioninclude tumor proteins, e.g., mutated oncogenes; viral proteinsassociated with tumors; and tumor mucins and glycolipids. The antigensmay be viral proteins associated with tumors would be those from theclasses of viruses noted above. Certain antigens may be characteristicof tumors (one subset being proteins not usually expressed by a tumorprecursor cell), or may be a protein which is normally expressed in atumor precursor cell, but having a mutation characteristic of a tumor.Other antigens include mutant variant(s) of the normal protein having analtered activity or subcellular distribution, e.g., mutations of genesgiving rise to tumor antigens.

Specific non-limiting examples of tumor antigens include: CEA, prostatespecific antigen (PSA), HER-2/neu, BAGE, GAGE, MAGE 1-4, 6 and 12, MUC(Mucin) (e.g., MUC-1, MUC-2, etc.), GM2 and GD2 gangliosides, ras, myc,tyrosinase, MART (melanoma antigen), Pmel 17(gp100), GnT-V intron Vsequence (N-acetylglucoaminyltransferase V intron V sequence), ProstateCa psm, PRAME (melanoma antigen), β-catenin, MUM-1-B (melanomaubiquitous mutated gene product), GAGE (melanoma antigen) 1, BAGE(melanoma antigen) 2-10, c-ERB2 (Her2/neu), EBNA (Epstein-Barr Virusnuclear antigen) 1-6, gp75, human papilloma virus (HPV) E6 and E7, p53,lung resistance protein (LRP), Bcl-2, and Ki-67. In addition, theimmunogenic molecule can be an autoantigen involved in the initiationand/or propagation of an autoimmune disease, the pathology of which islargely due to the activity of antibodies specific for a moleculeexpressed by the relevant target organ, tissue, or cells, e.g., SLE orMG. In such diseases, it can be desirable to direct an ongoingantibody-mediated (i.e., a Th2-type) immune response to the relevantautoantigen towards a cellular (i.e., a Th1-type) immune response.Alternatively, it can be desirable to prevent onset of or decrease thelevel of a Th2 response to the autoantigen in a subject not having, butwho is suspected of being susceptible to, the relevant autoimmunedisease by prophylactically inducing a Th1 response to the appropriateautoantigen. Autoantigens of interest include, without limitation: (a)with respect to SLE, the Smith protein, RNP ribonucleoprotein, and theSS-A and SS-B proteins; and (b) with respect to MG, the acetylcholinereceptor. Examples of other miscellaneous antigens involved in one ormore types of autoimmune response include, e.g., endogenous hormonessuch as luteinizing hormone, follicular stimulating hormone,testosterone, growth hormone, prolactin, and other hormones.

Antigens involved in autoimmune diseases, allergy, and graft rejectioncan be used in the compositions and methods of the invention. Forexample, an antigen involved in any one or more of the followingautoimmune diseases or disorders can be used in the present invention:diabetes, diabetes mellitus, arthritis (including rheumatoid arthritis,juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis),multiple sclerosis, myasthenia gravis, systemic lupus erythematosis,autoimmune thyroiditis, dermatitis (including atopic dermatitis andeczematous dermatitis), psoriasis, Sjogren's Syndrome, includingkeratoconjunctivitis sicca secondary to Sjogren's Syndrome, alopeciaareata, allergic responses due to arthropod bite reactions, Crohn'sdisease, aphthous ulcer, iritis, conjunctivitis, keratoconjunctivitis,ulcerative colitis, asthma, allergic asthma, cutaneous lupuserythematosus, scleroderma, vaginitis, proctitis, drug eruptions,leprosy reversal reactions, erythema nodosum leprosum, autoimmuneuveitis, allergic encephalomyelitis, acute necrotizing hemorrhagicencephalopathy, idiopathic bilateral progressive sensorineural hearingloss, aplastic anemia, pure red cell anemia, idiopathicthrombocytopenia, polychondritis, Wegener's granulomatosis, chronicactive hepatitis, Stevens-Johnson syndrome, idiopathic sprue, lichenplanus, Crohn's disease, Graves ophthalmopathy, sarcoidosis, primarybiliary cirrhosis, uveitis posterior, and interstitial lung fibrosis.Examples of antigens involved in autoimmune disease include glutamicacid decarboxylase 65 (GAD 65), native DNA, myelin basic protein, myelinproteolipid protein, acetylcholine receptor components, thyroglobulin,and the thyroid stimulating hormone (TSH) receptor. Examples of antigensinvolved in allergy include pollen antigens such as Japanese cedarpollen antigens, ragweed pollen antigens, rye grass pollen antigens,animal derived antigens such as dust mite antigens and feline antigens,histocompatiblity antigens, and penicillin and other therapeutic drugs.Examples of antigens involved in graft rejection include antigeniccomponents of the graft to be transplanted into the graft recipient suchas heart, lung, liver, pancreas, kidney, and neural graft components.The antigen may be an altered peptide ligand useful in treating anautoimmune disease.

As used herein, the term “epitope(s)” refer to a peptide or proteinantigen that includes a primary, secondary or tertiary structure similarto an epitope located within any of a number of pathogen polypeptidesencoded by the pathogen DNA or RNA. The level of similarity willgenerally be to such a degree that monoclonal or polyclonal antibodiesdirected against such polypeptides will also bind to, react with, orotherwise recognize, the peptide or protein antigen. Various immunoassaymethods may be employed in conjunction with such antibodies, such as,for example, Western blotting, ELISA, RIA, and the like, all of whichare known to those of skill in the art. The identification of pathogenepitopes, and/or their functional equivalents, suitable for use invaccines is part of the present invention. Once isolated and identified,one may readily obtain functional equivalents. For example, one mayemploy the methods of Hopp, as taught in U.S. Pat. No. 4,554,101,incorporated herein by reference, which teaches the identification andpreparation of epitopes from amino acid sequences on the basis ofhydrophilicity. The methods described in several other papers, andsoftware programs based thereon, can also be used to identify epitopiccore sequences (see, for example, Jameson and Wolf, 1988; Wolf et al.,1988; U.S. Pat. No. 4,554,101). The amino acid sequence of these“epitopic core sequences” may then be readily incorporated intopeptides, either through the application of peptide synthesis orrecombinant technology.

The preparation of vaccine compositions that includes the nucleic acidsthat encode antigens of the invention as the active ingredient, may beprepared as injectables, either as liquid solutions or suspensions;solid forms suitable for solution in, or suspension in, liquid prior toinfection can also be prepared. The preparation may be emulsified,encapsulated in liposomes. The active immunogenic ingredients are oftenmixed with carriers which are pharmaceutically acceptable and compatiblewith the active ingredient.

The term “pharmaceutically acceptable carrier” refers to a carrier thatdoes not cause an allergic reaction or other untoward effect in subjectsto whom it is administered. Suitable pharmaceutically acceptablecarriers include, for example, one or more of water, saline, phosphatebuffered saline, dextrose, glycerol, ethanol, or the like andcombinations thereof. In addition, if desired, the vaccine can containminor amounts of auxiliary substances such as wetting or emulsifyingagents, pH buffering agents, and/or adjuvants which enhance theeffectiveness of the vaccine. Examples of adjuvants that may beeffective include but are not limited to: aluminum hydroxide,N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine, MTP-PE and RIBI, whichcontains three components extracted from bacteria, monophosporyl lipidA, trehalose dimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2%squalene/Tween 80 emulsion. Other examples of adjuvants include DDA(dimethyldioctadecylammonium bromide), Freund's complete and incompleteadjuvants and QuilA. In addition, immune modulating substances such aslymphokines (e.g., IFN-γ, IL-2 and IL-12) or synthetic IFN-γ inducerssuch as poly I:C can be used in combination with adjuvants describedherein.

Pharmaceutical products that may include a naked polynucleotide with asingle or multiple copies of the specific nucleotide sequences that bindto specific DNA-binding sites of the apolipoproteins present on plasmalipoproteins as described in the current invention. The polynucleotidemay encode a biologically active peptide, antisense RNA, or ribozyme andwill be provided in a physiologically acceptable administrable form.Another pharmaceutical product that may spring from the currentinvention may include a highly purified plasma lipoprotein fraction,isolated according to the methodology, described herein from either thepatients blood or other source, and a polynucleotide containing singleor multiple copies of the specific nucleotide sequences that bind tospecific DNA-binding sites of the apolipoproteins present on plasmalipoproteins, prebound to the purified lipoprotein fraction in aphysiologically acceptable, administrable form.

Yet another pharmaceutical product may include a highly purified plasmalipoprotein fraction which contains recombinant apolipoprotein fragmentscontaining single or multiple copies of specific DNA-binding motifs,prebound to a polynucleotide containing single or multiple copies of thespecific nucleotide sequences, in a physiologically acceptableadministrable form. Yet another pharmaceutical product may include ahighly purified plasma lipoprotein fraction which contains recombinantapolipoprotein fragments containing single or multiple copies ofspecific DNA-binding motifs, prebound to a polynucleotide containingsingle or multiple copies of the specific nucleotide sequences, in aphysiologically acceptable administrable form.

The dosage to be administered depends to a great extent on the bodyweight and physical condition of the subject being treated as well asthe route of administration and frequency of treatment. A pharmaceuticalcomposition that includes the naked polynucleotide prebound to a highlypurified lipoprotein fraction may be administered in amounts rangingfrom 1 μg to 1 mg polynucleotide and 1 μg to 100 mg protein.

Administration of an rAb and rAb complexes a patient will follow generalprotocols for the administration of chemotherapeutics, taking intoaccount the toxicity, if any, of the vector. It is anticipated that thetreatment cycles would be repeated as necessary. It also is contemplatedthat various standard therapies, as well as surgical intervention, maybe applied in combination with the described gene therapy.

Where clinical application of a gene therapy is contemplated, it will benecessary to prepare the complex as a pharmaceutical compositionappropriate for the intended application. Generally this will entailpreparing a pharmaceutical composition that is essentially free ofpyrogens, as well as any other impurities that could be harmful tohumans or animals. One also will generally desire to employ appropriatesalts and buffers to render the complex stable and allow for complexuptake by target cells.

Aqueous compositions of the present invention may include an effectiveamount of the compound, dissolved or dispersed in a pharmaceuticallyacceptable carrier or aqueous medium. Such compositions can also bereferred to as inocula. The use of such media and agents forpharmaceutical active substances is well known in the art. Exceptinsofar as any conventional media or agent is incompatible with theactive ingredient, its use in the therapeutic compositions iscontemplated. Supplementary active ingredients also can be incorporatedinto the compositions. The compositions of the present invention mayinclude classic pharmaceutical preparations. Dispersions also can beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

Disease States. Depending on the particular disease to be treated,administration of therapeutic compositions according to the presentinvention will be via any common route so long as the target tissue isavailable via that route in order to maximize the delivery of antigen toa site for maximum (or in some cases minimum) immune response.Administration will generally be by orthotopic, intradermal,subcutaneous, intramuscular, intraperitoneal or intravenous injection.Other areas for delivery include: oral, nasal, buccal, rectal, vaginalor topical. Topical administration would be particularly advantageousfor treatment of skin cancers. Such compositions would normally beadministered as pharmaceutically acceptable compositions that includephysiologically acceptable carriers, buffers or other excipients.

Vaccine or treatment compositions of the invention may be administeredparenterally, by injection, for example, either subcutaneously orintramuscularly. Additional formulations which are suitable for othermodes of administration include suppositories, and in some cases, oralformulations or formulations suitable for distribution as aerosols. Inthe case of the oral formulations, the manipulation of T-cell subsetsemploying adjuvants, antigen packaging, or the addition of individualcytokines to various formulation that result in improved oral vaccineswith optimized immune responses. For suppositories, traditional bindersand carriers may include, for example, polyalkylene glycols ortriglycerides; such suppositories may be formed from mixtures containingthe active ingredient in the range of 0.5% to 10%, preferably 1%-2%.Oral formulations include such normally employed excipients as, forexample, pharmaceutical grades of mannitol, lactose, starch magnesiumstearate, sodium saccharine, cellulose, magnesium carbonate, and thelike. These compositions take the form of solutions, suspensions,tablets, pills, capsules, sustained release formulations or powders andcontain 10%-95% of active ingredient, preferably 25-70%.

The antigen encoding nucleic acids of the invention may be formulatedinto the vaccine or treatment compositions as neutral or salt forms.Pharmaceutically acceptable salts include the acid addition salts(formed with free amino groups of the peptide) and which are formed withinorganic acids such as, for example, hydrochloric or phosphoric acids,or with organic acids such as acetic, oxalic, tartaric, maleic, and thelike. Salts formed with the free carboxyl groups can also be derivedfrom inorganic bases such as, for example, sodium, potassium, ammonium,calcium, or ferric hydroides, and such organic bases as isopropylamine,trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

Vaccine or treatment compositions are administered in a mannercompatible with the dosage formulation, and in such amount as will beprophylactically and/or therapeutically effective. The quantity to beadministered depends on the subject to be treated, including, e.g.,capacity of the subject's immune system to synthesize antibodies, andthe degree of protection or treatment desired. Suitable dosage rangesare of the order of several hundred micrograms active ingredient pervaccination with a range from about 0.1 mg to 1000 mg, such as in therange from about 1 mg to 300 mg, and preferably in the range from about10 mg to 50 mg. Suitable regiments for initial administration andbooster shots are also variable but are typified by an initialadministration followed by subsequent inoculations or otheradministrations. Precise amounts of active ingredient required to beadministered depend on the judgment of the practitioner and may bepeculiar to each subject. It will be apparent to those of skill in theart that the therapeutically effective amount of nucleic acid moleculeor fusion polypeptides of this invention will depend, inter alia, uponthe administration schedule, the unit dose of antigen administered,whether the nucleic acid molecule or fusion polypeptide is administeredin combination with other therapeutic agents, the immune status andhealth of the recipient, and the therapeutic activity of the particularnucleic acid molecule or fusion polypeptide.

The compositions can be given in a single dose schedule or in a multipledose schedule. A multiple dose schedule is one in which a primary courseof vaccination may include, e.g., 1-10 separate doses, followed by otherdoses given at subsequent time intervals required to maintain and orreinforce the immune response, for example, at 1-4 months for a seconddose, and if needed, a subsequent dose(s) after several months. Periodicboosters at intervals of 1-5 years, usually 3 years, are desirable tomaintain the desired levels of protective immunity. The course of theimmunization can be followed by in vitro proliferation assays ofperipheral blood lymphocytes (PBLs) co-cultured with ESAT6 or ST-CF, andby measuring the levels of IFN-γ released from the primed lymphocytes.The assays may be performed using conventional labels, such asradionucleotides, enzymes, fluorescent labels and the like. Thesetechniques are known to one skilled in the art and can be found in U.S.Pat. Nos. 3,791,932, 4,174,384 and 3,949,064, relevant portionsincorporated by reference.

The modular rAb carrier and/or conjugated rAb carrier-(cohesion/dockerinand/or dockerin-cohesin)-antigen complex (rAb-DC/DC-antigen vaccine) maybe provided in one or more “unit doses” depending on whether the nucleicacid vectors are used, the final purified proteins, or the final vaccineform is used. Unit dose is defined as containing apredetermined-quantity of the therapeutic composition calculated toproduce the desired responses in association with its administration,i.e., the appropriate route and treatment regimen. The quantity to beadministered, and the particular route and formulation, are within theskill of those in the clinical arts. The subject to be treated may alsobe evaluated, in particular, the state of the subject's immune systemand the protection desired. A unit dose need not be administered as asingle injection but may include continuous infusion over a set periodof time. Unit dose of the present invention may conveniently may bedescribed in terms of DNA/kg (or protein/Kg) body weight, with rangesbetween about 0.05, 0.10, 0.15, 0.20, 0.25, 0.5, 1, 10, 50, 100, 1,000or more mg/DNA or protein/kg body weight are administered. Likewise theamount of rAb-DC/DC-antigen vaccine delivered can vary from about 0.2 toabout 8.0 mg/kg body weight. Thus, in particular embodiments, 0.4 mg,0.5 mg, 0.8 mg, 1.0 mg, 1.5 mg, 2.0 mg, 2.5 mg, 3.0 mg, 4.0 mg, 5.0 mg,5.5 mg, 6.0 mg, 6.5 mg, 7.0 mg and 7.5 mg of the vaccine may bedelivered to an individual in vivo. The dosage of rAb-DC/DC-antigenvaccine to be administered depends to a great extent on the weight andphysical condition of the subject being treated as well as the route ofadministration and the frequency of treatment. A pharmaceuticalcomposition that includes a naked polynucleotide prebound to a liposomalor viral delivery vector may be administered in amounts ranging from 1μg to 1 mg polynucleotide to 1 μg to 100 mg protein. Thus, particularcompositions may include between about 1 μg, 5 μg, 10 μg, 20μg, 30 μg,40 μg, 50 μg, 60 μg, 70 μg, 80 μg, 100 μg, 150 μg, 200 μg, 250 μg, 500μg, 600 μg, 700 μg, 800 μg, 900 μg or 1,000 μg polynucleotide or proteinthat is bound independently to 1 μg, 5 μg, 10 μg, 20 μg, 3.0 μg, 40 μg50 μg, 60 μg, 70 μg, 80 μg, 100 μg, 150 μg, 200 μg, 250 μg, 500 μg, 600μg, 700 μg, 800 μg, 900 μg, 1 mg, 1.5 mg, 5 mg, 10 mg, 20 mg, 30 mg, 40mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg or 100 mg vector.

The present invention was tested in an in vitro cellular system thatmeasures immune stimulation of human Flu-specific T cells by dendriticcells to which Flu antigen has been targeted. The results shown hereindemonstrate the specific expansion of such antigen specific cells atdoses of the antigen which are by themselves ineffective in this system.

The present invention may also be used to make a modular rAb carrierthat is, e.g., a recombinant humanized mAb (directed to a specific humandendritic cell receptor) complexed with protective antigens from Ricin,Anthrax toxin, and Staphylococcus B enterotoxin. The potential marketfor this entity is vaccination of all military personnel and storedvaccine held in reserve to administer to large population centers inresponse to any biothreat related to these agents. The invention hasbroad application to the design of vaccines in general, both for humanand animal use. Industries of interest include the pharmaceutical andbiotechnology industries.

The present invention includes compositions and methods, includingvaccines, that specifically target (deliver) antigens toantigen-presenting cells (APCs) for the purpose of eliciting potent andbroad immune responses directed against the antigen. These compositionsevoke protective or therapeutic immune responses against the agent(pathogen or cancer) from which the antigen was derived. In addition theinvention creates agents that are directly, or in concert with otheragents, therapeutic through their specific engagement of a receptorcalled DC-ASGPR that is expressed on antigen-presenting cells.

The novel recombinant humanized mAb (directed to the specific humandendritic cell receptor DC-ASGPR) fused through the antibody (Ab) heavychain to antigens known or suspected to encode protective antigens.These include as examples for vaccination against variousagents—hemagglutinins from Influenza H5N1; HIV gag from attenuatedtoxins from Ricin, Anthrax toxin, and Staphylococcus B enterotoxin;‘strings’ of antigenic peptides from melanona antigens, etc. The presentinvention may be used as a preventative or therapeutic vaccination forat risk or infected patients. The invention has broad application forvaccination against many diseases and cancers, both for human and animaluse. Industries that can use the present invention include thepharmaceutical and biotechnological.

The present invention can be used to target antigens to APC forvaccination purposes. It is not known which antigen internalizingreceptor will be best suited for this purpose. The invention describesparticularly advantageous features of DC-ASGPR as for this purpose.Furthermore, the invention shows that engaging DC-ASGPR can bebeneficial in the sense of activating the immune system with highlypredicted significant therapeutic benefit.

The present invention includes the development of high affinitymonoclonal antibodies against human DC-ASGPR. Receptor ectodomain.hIgG(human IgG1Fc) and AP (human placental alkaline phosphatase) fusionproteins were produced for immunization of mice and screening of mAbs,respectively. An expression construct for hDCIR ectodomain.IgG wasdescribed previously (Bates, Fournier et al. 1999) and used the mouseSLAM (mSLAM) signal peptide to direct secretion (Bendtsen, Nielsen etal. 2004). An expression vector for hDCIR ectodomain.AP was generatedusing PCR to amplify AP resides 133-1581 (gbIBC0096471) while adding aproximal in-frame Xho I site and a distal TGA stop codon and Not I site.This Xho I-Not I fragment replaced the IgG coding sequence in the abovehDCIR ectodomain.IgG vector. DC-ASGPR ectodomain constructs in the sameIg and AP vector series contained inserts encoding (bp 484-1251,gi153832017). DC-ASGPR fusion proteins were produced using theFreeStyle™ 293 Expression System (Invitrogen) according to themanufacturer's protocol (1 mg total plasmid DNA with 1.3 ml 293 Fectinreagent/L of transfection). For rAb production, equal amounts of vectorencoding the H and L chain were co-transfected. Transfected cells arecultured for 3 days, the culture supernatant was harvested and freshmedia added with continued incubation for two days. The pooledsupernatants were clarified by filtration. Receptor ectodomain.hIgG waspurified by HiTrap protein A affinity chromatography with elution by 0.1M glycine pH 2.7 and then dialyzed versus PBS. rAbs (recombinantantibodies described later) were purified similarly, by using HiTrapMabSelect™ columns. Mouse mAbs were generated by conventional cellfusion technology. Briefly, 6-week-old BALB/c mice were immunizedintraperitonealy with 20 μg of receptor ectodomain.hIgGFc fusion proteinwith Ribi adjuvant, then boosts with 20 μg antigen 10 days and 15 dayslater. After 3 months, the mice were boosted again three days prior totaking the spleens. Alternately, mice were injected in the footpad with1-10 μg antigen in Ribi adjuvant every 3-4 days over a 30-40 day period.3-4 days after a final boost, draining lymph nodes were harvested. Bcells from spleen or lymph node cells were fused with SP2/O-Ag 14 cells(Shulman, Wilde et al. 1978) using conventional techniques.

ELISA was used to screen hybridoma supernatants against the receptorectodomain fusion protein compared to the fusion partner alone, orversus the receptor ectodomain fused to AP (Bates, Fournier et al.1999). Positive wells were then screened in FACS using 293F cellstransiently transfected with expression plasmids encoding full-lengthreceptor cDNAs. Selected hybridomas were single cell cloned and expandedin CELLine flasks (Intergra). Hybridoma supernatants were mixed with anequal volume of 1.5 M glycine, 3 M NaCl, 1×PBS, pH 7.8 and tumbled withMabSelect resin. The resin was washed with binding buffer and elutedwith 0.1 M glycine, pH 2.7. Following neutralization with 2 M Tris, mAbswere dialyzed versus PBS.

Characterization of purified anti-DC-ASGPR monoclonal antibodies bydirect ELISA. the relative affinities of several anti-DC-ASGPR mAbs byELISA were determined (i.e., DC-ASGPR.Ig protein is immobilized on themicroplate surface and the antibodies are tested in a dose titrationseries for their ability to bind to DC-ASGPR.Ig (as detected by ananti-mouse IgG.HRP conjugate reagent. In this example, PAB42 and PAB44show higher affinity binding than other mAbs. The same mAbs fail to bindsignificantly to human Ig bound to the microplate surface. This showsthat the mAbs react to the DC-ASGPR ectodomain part of the DC-ASGPR.Igfusion protein (data not shown).

Characterization of purified anti-DC-ASGPR monoclonal antibodies byindirect ELISA. Next, the relative affinities of several anti-DC-ASGPRmAbs were determined by ELISA (i.e., anti-DC-ASGPR mAb is immobilized onthe microplate surface and tested in a dose titration series for theirability to bind to DC-ASGPR.AP reagent. It was found that thesupernatants from the hybridomas listed as: PAB42, PAB44 and PAB54 showhigher affinity binding than other mAbs (data not shown).

Characterization of anti-DC-ASGPR mAbs by FACS. The panel of mAbs wasalso tested by FACS versus 293F cells transfected with expressionplasmid directing synthesis of cell surface DC-ASGPR. Mean fluorescenceintensity of the signal was subtracted from the analogous signal versusnon-transfected 293F cells. By this criterion, the mAbs are able to bindto specifically to the surface of cells bearing DC-ASGPR. Some mAbs,e.g., 37A7 appear particularly advantageous in this regard (data notshown).

FIGS. 1A to 1D shows that signaling through DC-ASGPR activates DCs. DCsare the primary immune cells that determine the results of immuneresponses, either induction or tolerance, depending on their activation(15). The role of LLRs in DC activation is not clear yet. Therefore, wetested whether triggering the LLR DC-ASGPR can result in the activationof DCs. Both three and six day in vitro cultured GM/IL-4 DCs expressLOX-1, ASGPR, and CLEC-6 (FIG. 1A). Six day DCs were stimulated with mAbspecific to DC-ASGPR, and data in FIG. 1B show that signals throughDC-ASGPR could activate DCs, resulting in the increased expression ofCD86 and HLA-DR. Triggering DC-ASGPR on DCs also resulted in theincreased production of IL-6, MCP-1, IL-12p40, and IL-8 from DCs (FIG.1C). Other cytokines and chemokines, TNFa, IP-10, MIP-1α, and IL-10,were also significantly increased (data not shown) by signaling throughDC-ASGPR, suggesting that DC-ASGPR can deliver cellular signals toactivate DCs. Consistently, DCs stimulated with DC-ASGPR specific mAbexpressed increased levels of multiple genes, including co-stimulatorymolecules as well as chemokine and cytokine-related genes (FIG. 1D). Thepossible contribution of LLRs in TLR2 and TLR4-mediated immune cellactivation has been described previously (13, 16). We observed thatsignals through DC-ASGPR could synergize with signal through CD40 for afurther activation of DCs (FIG. 1E). This is important because LLRscould serve as co-stimulatory molecules during in vivo DC activation.Taken together, data in FIG. 1 prove that signaling through DC-ASGPR canactivate DCs and that DC-ASGPR serves as a co-stimulatory molecule forthe activation of DCs. DC-ASGPR engagement during CD40-CD40L interactionresults in dramatically increased production of IL-12p70.

DCs stimulated through DC-ASGPR induce potent humoral immune responses.DCs play an important role in humoral immune responses by providingsignals for both T-dependent and T-independent B cell responses (19-22)and by transferring antigens to B cells (23, 24). In addition to DCs,signaling through TLR9 as a third signal is necessary for efficient Bcell responses (25, 26).

Therefore, we tested the role of DC-ASGPR in DCs-mediated humoral immuneresponses in the presence of TLR9 ligand, CpG. Six day GM/IL-4 DCs werestimulated with anti-DC-ASGPR mAb, and then purified B cells wereco-cultured. As shown in FIG. 2A, DCs activated with anti-DC-ASGPR mAbresulted in remarkably enhanced B cell proliferation (CFSE dilution) andplasma cell differentiation (CD38⁺CD20⁻), compared to DCs stimulatedwith control mAb. CD38⁺CD20⁻ B cells have a typical morphology of plasmacells, but they do not express CD138. The majority of proliferatingcells did not express CCR2, CCR4, CCR6, or CCR7. The amounts of totalimmunoglobulins (Igs) produced were measured by ELISA (FIG. 2B).Consistent with the data in FIG. 2A, B cells cultured withanti-DC-ASGPR-stimulated DCs resulted in significantly increasedproduction of total IgM, IgG, and IgA. In addition to the total Igs, wealso observed that DCs activated by triggering DC-ASGPR are more potentthan DCs stimulated with control mAb for the production ofinfluenza-virus-specific IgM, IgG, and IgA (FIG. 2C) by B cells,suggesting that DC-ASGPR-mediated DC activation contributes to bothtotal and antigen specific humoral immune responses. We tested the roleof DC-ASGPR in ex vivo antigen presenting cells (APCs) in humoral immuneresponses. Parts of APCs in PBMCs, including CD19⁺ and CD14⁺ cells,express DC-ASGPR (Supplementary FIG. 2). PBMCs from buffy coats werecultured in the plates coated with anti-DC-ASGPR mAb, and the total Igsand B cell proliferation were measured. Consistent with the datagenerated from DCs (FIG. 2A), APCs stimulated through DC-ASGPR resultedin enhanced B cell proliferation and plasma cell differentiation in theabsence (upper panels in FIG. 2d ) or presence (lower panels in FIG. 2D)of TLR9 ligand. The total IgM, IgG, and IgA were also significantlyincreased when PBMCs were cultured in the plates coated with mAb againstDC-ASGPR (FIG. 2e ). As shown in FIG. 1, DCs activated by signalingthrough DC-ASGPR have matured phenotypes and produce large amounts ofinflammatory cytokines and chemokines, and both matured DC phenotypesand soluble factors from DCs could contribute to the enhanced B cellsresponses (FIG. 2). However, DC-derived B lymphocyte stimulator protein(BLyS, BAFF) and a proliferation-inducing ligand (APRIL) are alsoimportant molecules by which DCs can directly regulate human B cellproliferation and function (27-30). Therefore, we tested whether signalsthrough DC-ASGPR could alter the expression levels of BLyS and APRIL.Data in FIG. 2d show that DCs stimulated through DC-ASGPR expressedincreased levels of intracellular APRIL as well as APRIL secreted, butnot BLyS (not shown). Expression levels of BLyS and APRIL receptors on Bcells in the mixed cultures were measured, but there was no significantchange (not shown).

DC-ASGPR contributes to B cell activation and Ig production. CD19⁺ Bcells express DC-ASGPR (FIG. 3A). Therefore, we tested the role ofDC-ASGPR in B cell activation. Data in FIG. 3B show that B cellsstimulated through DC-ASGPR produced significantly higher amounts ofchemokines. In addition to IL-8 and MIP-1α, slight increases in IL-6 andTNFα were also observed when B cells were stimulated with theanti-DC-ASGPR mAb, compared to control mAb. Genes related to cellactivation were also up-regulated (FIG. 3C). B cells produced IgM, IgG,and IgA when they were stimulated through DC-ASGPR (FIG. 3D), suggestingthat DC-ASGPR could play an important role in the maintenance of normalimmunoglobulin levels in vivo. However, signaling through DC-ASGPR alonedid not induce significant B cell proliferation.

Role of DC-ASGPR in T cell responses. DCs stimulated through DC-ASGPRexpress enhanced levels of co-stimulatory molecules and produceincreased amounts of cytokines and chemokines (see FIG. 1), suggestingthat DC-ASGPR contributes to cellular immune responses as well ashumoral immune responses. This was tested by a mixed lymphocyte reaction(MLR). Proliferation of purified allogeneic T cells was significantlyenhanced by DCs stimulated with mAb specific for DC-ASGPR (FIG. 4A). DCsactivated through DC-ASGPR could also prime Mart-1-specific CD8 T cellsmore efficiently than DC stimulated with control mAb (upper panels inFIG. 4B). More importantly, signaling through DC-ASGPR permitted DCs tocross-prime Mart-1 peptides to CD8 T cells (lower panels in FIG. 4B).This indicates that DC-ASGPR plays an important role in enhancing DCfunction, resulting in better priming and cross-priming of antigens toCD8 T cells. The role of DC-ASGPR expressed on the mixture of APCs inPBMCs in activation of T cell responses is shown in FIG. 4C where PBMCsstimulated with mAb to DC-ASGPR resulted in an increased frequency ofFlu M1 tetramer specific CD8 T cells compared to DCs stimulated withcontrol mAb. This enhanced antigen specific CD8 T cell response wassupported by the data in FIG. 4D, showing that DCs stimulated throughDC-ASGPR significantly increase CD4 T cell proliferation.

Antibodies and tetramers—Antibodies (Abs) for surface staining of DCsand B cells, including isotype control Abs, were purchased from BDBiosciences (CA). Abs for ELISA were purchased from Bethyl (TX).Anti-BLyS and anti-APRIL were from PeproTech (NJ). Tetramers,HLA-A*0201-GILGFVFTL (SEQ ID NO.: 1) (Flu M1) and HLA-A*0201-ELAGIGILTV(SEQ ID NO.: 2) (Mart-1), were purchased from Beckman Coulter (CA).

Cells and cultures—Monocytes (1×10⁶/m1) from normal donors were culturedin Cellgenics (France) media containing GM-CSF (100 ng/ml) and IL-4 (50ng/ml) (R&D, CA). For day three and day six, DCs, the same amounts ofcytokines were supplemented into the media on day one and day three,respectively. B cells were purified with a negative isolation kit (BD).CD4 and CD8 T cells were purified with magnetic beads coated withanti-CD4 or CD8 (Milteniy, Calif.). PBMCs were isolated from Buffy coatsusing Percoll™ gradients (GE Healthcare UK Ltd, Buckinghamshire, UK) bydensity gradient centrifugation. For DC activation, 1×10⁵ DCs werecultured in the mAb-coated 96-well plate for 16-18 h. mAbs (1-2 μg/well)in carbonate buffer, pH 9.4, were incubated for at least 3 h at 37° C.Culture supernatants were harvested and cytokines/chemokines weremeasured by Luminex (Biorad, Calif.). For gene analysis, DCs werecultured in the plates coated with mAbs for 8 h. In some experiments,soluble 50 ng/ml of CD40L (R&D, CA) or 50 nM CpG (InVivogen, CA) wasadded into the cultures. In the DCs and B cell co-cultures, 5×10³ DCsresuspended in RPMI 1640 with 10% FCS and antibiotics (Biosource, CA)were first cultured in the plates coated with mAbs for at least 6 h, andthen 1×10⁵ purified autologous B cells labeled with CFSE (MolecularProbes, OR) were added. In some experiments, DCs were pulsed with 5 moi(multiplicity of infection) of heat-inactivated influenza virus (A/PR/8H1N1) for 2 h, and then mixed with B cells. For the DCs and T cellco-cultures, 5×10³ DCs were cultured with 1×10⁵ purified autologous CD8T cells or mixed allogeneic T cells. Allogeneic T cells were pulsed with1 μCi/well ³[H]-thymidine for the final 18 h of incubation, and then cpmwere measured by a μ-counter (Wallac, Minn.). 5×10⁵ PBMCs/well werecultured in the plates coated with mAbs. The frequency of Mart-1 and FluM1 specific CD8 T cells was measured by staining cells with anti-CD8 andtetramers on day ten and day seven of the cultures, respectively. 10 μMof Mart-1 peptide (ELAGIGILTV) (SEQ ID NO.: 2) and 20 nM of recombinantprotein containing Mart-1 peptides (see below) were added to the DC andCD8 T cell cultures. 20 nM purified recombinant Flu M1 protein (seebelow) was add to the PBMC cultures.

Monoclonal antibodies—Mouse mAbs were generated by conventionaltechnology. Briefly, six-week-old BALB/c mice were immunized i.p. with20 μg of receptor ectodomain.hIgGFc fusion protein with Ribi adjuvant,then boosts with 20 μg antigen ten days and fifteen days later. Afterthree months, the mice were boosted again three days prior to taking thespleens. Alternately, mice were injected in the footpad with 1-10 μgantigen in Ribi adjuvant every three to four days over a thirty to fortyday period. Three to four days after a final boost, draining lymph nodeswere harvested. B cells from spleen or lymph node cells were fused withSP2/O-Ag 14 cells. Hybridoma supernatants were screened to analyze Absto the receptor ectodomain fusion protein compared to the fusion partneralone, or the receptor ectodomain fused to alkaline phosphatase (44).Positive wells were then screened in FACS using 293F cells transientlytransfected with expression plasmids encoding full-length receptorcDNAs. Selected hybridomas were single cell cloned and expanded inCELLine flasks (Integra, CA). Hybridoma supernatants were mixed with anequal volume of 1.5 M glycine, 3 M NaCl, 1×PBS, pH 7.8 and tumbled withMabSelect resin. The resin was washed with binding buffer and elutedwith 0.1 M glycine, pH 2.7. Following neutralization with 2 M Tris, mAbswere dialyzed versus PBS.

ELISA—Sandwich ELISA was performed to measure total IgM, IgG, and IgA aswell as flu-specific immunoglobulins (Igs). Standard human serum(Bethyl) containing known amounts of Igs and human AB serum were used asstandard for total Igs and flu-specific Igs, respectively. Flu specificAb titers, units, in samples were defined as dilution factor of AB serumthat shows an identical optical density. The amounts of BAFF and BLySwere measured by ELISA kits (Bender MedSystem, CA).

RNA purification and gene analysis—Total RNA extracted with RNeasycolumns (Qiagen), and analyzed with the 2100 Bioanalyser (Agilent).Biotin-labeled cRNA targets were prepared using the Illumina totalpreplabeling kit (Ambion) and hybridized to Sentrix Human6 BeadChips (46Ktranscripts). These microarrays consist of 50mer oligonucleotide probesattached to 3 μm beads which are lodged into microwells etched at thesurface of a silicon wafer. After staining with Streptavidin-Cy3, thearray surface is imaged using a sub-micron resolution scannermanufactured by Illumina (Beadstation 500X). A gene expression analysissoftware program, GeneSpring, Version 7.1 (Agilent), was used to performdata analysis.

Expression and purification of recombinant Flu M1 and MART-1proteins—PCR was used to amplify the ORF of Influenza A/PuertoRico/8/34/Mount Sinai (H1N1) M1 gene while incorporating an Nhe I sitedistal to the initiator codon and a Not I site distal to the stop codon.The digested fragment was cloned into pET-28b(+) (Novagen), placing theM1 ORF in-frame with a His6 tag, thus encoding His.Flu M1 protein. ApET28b (+) derivative encoding an N-terminal 169 residue cohesin domainfrom C. thermocellum (unpublished) inserted between the Nco I and Nhe Isites expressed Coh.His. For expression ofCohesin-Flex-hMART-1-PeptideA-His, the sequenceGACACCACCGAGGCCCGCCACCCCCACCCCCCCGTGACCACCCCCACCACCACCGACCGGAAGGGCACCACCGCCGAGGAGCTGGCCGGCATCGGCATCCTGACCGTGATCCTGGGCGGCAAGCGGACCAACAACAGCACCCCCACCAAGGGCGAATTCTGCAGATATCCATCACACTGGCGGCCG (SEQ ID NO.: 3) (encodingDTTEARHPHPPVTTPTTDRKGTTAEELAGIGILTVILGGKRTNNSTPTKGEFCRYPSHWRP (SEQ IDNO.: 4)—the marked residues are the immunodominant HLA-A2-restrictedpeptide and the underlined residues surrounding the peptide are fromMART-1) was inserted between the Nhe I and Xho I sites of the abovevector. The proteins were expressed in E. coli strain BL21 (DE3)(Novagen) or T7 Express (NEB), grown in LB at 37° C. with selection forkanamycin resistance (40 μg/ml) and shaking at 200 rounds/min to mid logphase growth when 120 mg/L IPTG was added. After three hours, the cellswere harvested by centrifugation and stored at −80° C. E. coli cellsfrom each 1 L fermentation were resuspended in 30 ml ice-cold 50 mMTris, 1 mM EDTA pH 8.0 (buffer B) with 0.1 ml of protease inhibitorCocktail II (Calbiochem, CA). The cells were sonicated on ice 2×5 min atsetting 18 (Fisher Sonic Dismembrator 60) with a 5 min rest period andthen spun at 17,000 r.p.m. (Sorvall SA-600) for 20 min at 4° C. ForHis.Flu M1 purification the 50 ml cell lysate supernatant fraction waspassed through 5 ml Q Sepharose beads and 6.25 ml 160 mM Tris, 40 mMimidazole, 4 M NaCl pH 7.9 was added to the Q Sepharose flow through.This was loaded at 4 ml/min onto a 5 ml HiTrap chelating HP columncharged with Ni++. The column-bound protein was washed with 20 mM NaPO₄,300 mM NaCl pH 7.6 (buffer D) followed by another wash with 100 mMH₃COONa pH 4.0. Bound protein was eluted with 100 mM H₃COONa pH 4.0. Thepeak fractions were pooled and loaded at 4 ml/min onto a 5 ml HiTrap Scolumn equilibrated with 100 mM H₃COONa pH 5.5, and washed with theequilibration buffer followed by elution with a gradient from 0-1 M NaClin 50 mM NaPO₄ pH 5.5. Peak fractions eluting at about 500 mM NaCl werepooled. For Coh.Flu M1.His purification, cells from 2 L of culture werelysed as above. After centrifugation, 2.5 ml of Triton X114 was added tothe supernatant with incubation on ice for 5 min. After furtherincubation at 25° C. for 5 min, the supernatant was separated from theTriton X114 following centrifugation at 25° C. The extraction wasrepeated and the supernatant was passed through 5 ml of Q Sepharosebeads and 6.25 ml 160 mM Tris, 40 mM imidazole, 4 M NaCl pH 7.9 wasadded to the Q Sepharose flow through. The protein was then purified byNi⁺⁺ chelating chromatography as described above and eluted with 0-500mM imidazole in buffer D.

Only particular anti-DC-ASGPR mAbs have DC activation properties—Theinvention discloses that DC activation is not a general property ofanti-DC-ASGPR antibodies, rather only certain anti-DC-ASGPR mAbs havethis function. FIG. 5 shows that only certain mAbs activate DCS throughthe DC-ASGPR, which must be characterized by screening against actualDCs.

Particular sequences corresponding to the L and H variable regions ofanti-DC-ASGPR mAbs—The invention encompasses particular amino acidsequences shown below corresponding to anti-DC-ASGPR monoclonalantibodies that are desirable components (in the context of e.g.,humanized recombinant antibodies) of therapeutic or protective products.The following are such sequences in the context of chimeric mouse Vregion—human C region recombinant antibodies.

[mAnti-ASGPR_49C11_7H-LV-hIgG4H-C] is (SEQ ID NO.: 5)DVQLQESGPDLVKPSQSLSLTCTVTGYSITSGYSWHWIRQFPGNKLEWMGYILFSGSTNYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYFCARSNYGSFASWGQGTLVTVSAAKTTGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKAS.The above sequence is a chimera between the H chain V-region of the mAb49C11 (shown underlined) and the C region of hIgG4.

[mAnti-ASGPR_49C11_7K-LV-hIgGK-C] is the corresponding L chain chimera -(SEQ ID NO.: 6)QIVLTQSPAIMSASPGEKVTMTCSASSSVSHMHWYQQKSGTSPKRWIYDTSRLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSHPWSFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.[mAnti-ASGPR_4G2.2_Hv-V-hIgG4H-C] is - (SEQ ID NO.: 7)QIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNWVKQVPGKGLRWMGWMDTFTGEPTYADDFKGRFAFSLETSASTAYLQINSLKNEDTATYFCARGGILRLNYFDYWGQGTTLTVSSAKTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKAS.[mAnti-ASGPR_4G2.2_Kv-V-hIgGK-C] is - (SEQ ID NO.: 8)DIQMTQSSSSFSVSLGDRVTITCKASEDIYNRLGWYQQKPGNAPRLLISGATSLETGVPSRFSGSGSGKDYALSITSLQTEDLATYYCQQCWTSPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC. [mAnti-ASGPR_5F10H-LV-hIgG4H-C] is -(SEQ ID NO.: 9)EVQLQQSGPELVKPGASVKMSCKASGYTFTDYYMKWVKQSHGKSLEWIGDINPNYGDTFYNQKFEGKATLTVDKSSRTAYMQLNSLTSEDSAVYYCGRGDYGYFDVWGAGTTVTVSSAKTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS RLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLLSLGKAS. [mAnti-ASGPR_5F10K-LV-hIgGK-C] is -(SEQ ID NO.: 10)DIVMTQSHKFMSTSVGDRVSITCKASQDVGTAVAWYQQKPGQSPKLLIYWASTRHTGVPDRFTGSSGTDFTLTINNVQSEDLADYFCQQYSSNPYMFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.[mAnti-ASGPR1H11H-V-hIgG4H-C] is - (SEQ ID NO.: 11)QLQQSGPELVKPGASVKISCKTSGYTFTEYTMHWVRQSHGKSLEWIGGINPINGGPTYNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCARWDYGSRDVMDYWGQGTSVTVSSAKTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKAS. [mAnti-ASGPR1H11K-LV-hIgGK-C] is -(SEQ ID NO.: 12)NIVMTQSPKSMSMSVGERVTLSCKASENVGTYVSWYQQRPEQSPKLLIYGASNRYTGVPDRFTGSGSATDFTLTISSVQAEDLADYHCGQTYSYIFTFGSGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.The invention envisions these V-region sequences and related sequencesmodified by those well versed in the art to e.g., enhance affinity forDC-ASGPR and/or integrated into human V-region framework sequences to beengineered into expression vectors to direct the expression of proteinforms that can bind to DC-ASGPR on antigen presenting cells.

Engineered recombinant anti-DC-ASGPR recombinant antibody—antigen fusionproteins ((rAb.antigen) are efficacious prototype vaccines invitro—Expression vectors can be constructed with diverse protein codingsequence e.g., fused in-frame to the H chain coding sequence. Forexample, antigens such as Influenza HA5, Influenza M1, HIV gag, orimmuno-dominant peptides from cancer antigens, or cytokines, can beexpressed subsequently as rAb.antigen or rAb.cytokine fusion proteins,which in the context of this invention, can have utility derived fromusing the anti-DC-ASGPR V-region sequence to bring the antigen orcytokine (or toxin) directly to the surface of the antigen presentingcell bearing DC-ASGPR. This permits internalization of e.g.,antigen—sometimes associated with activation of the receptor and ensuinginitiation of therapeutic or protective action (e.g., via initiation ofa potent immune response, or via killing of the targeted cell. Anexemplative prototype vaccine based on this concept could use a H chainvector such as

[mAnti-ASGPR_5F10H-LV-hIgG4H-C-Flex-F1uHA5-1- 6xH] or - (SEQ ID NO.: 13)EVQLQQSGPELVKPGASVKMSCKASGYTFTDYYMKWVKQSHGKSLEWIGDINPNYGDTFYNQKFEGKATLTVDKSSRTAYMQLNSLTSEDSAVYYCGRGDYGYFDVWGAGTTVTVSSAKTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKASDTTEPATPTTPVT TDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKKHNGKLCDLDGVKPLILRDCSVAGWLLGNPMCDEFINVPEWSVIVEKANPVNDLCYPGDFNDYEELKHLLSRINHFEKIQIIPKSSWSSHEASLGVSSACPYQGKSSFFRNVVWLIKKNSTYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTKLYQNPTTYISVGTSTLNQRLVPRIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSTIMKSELEYGNCNTKCQTPMGAINSSMPFHNIHPLTIGECPKY VKSNRLVLAHHHHHH.The above sequence corresponds to the chimeric H chain shown alreadyfused via a flexible linker sequence (shown italicized) to HA-1 domainof avian Flu HA5 (shown in bold). This can be co-expressed with thecorresponding L chain chimeric sequence already shown above. Similarly,the sequence

[mAnti-ASGPR_49C11_7H-LV-hIgG4H-C-Dockerin]  (SEQ ID NO.: 14)DVQLQESGPDLVKPSQSLSLTCTVTGYSITSGYSWHWIRQFPGNKLEWMGYILFSGSTNYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYFCARSNYGSFASWGQGTLVTVSAAKTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKASNSPQNEVLYGDVNDDGKVNSTDLTLLKRYVLKAVSTLPSSKAEKNADVNRDGRVNSSDVTILSR YLIRVIEKLPIcan be used to express via co-transfection of the corresponding L chainsequence already shown above a rAb.Dockerin fusion protein.

FIG. 5 shows that certain anti-DC-ASGPR mAbs can activate DC.GM-CSF/IL-4. DC were incubated for 24 hrs with one of a panel of 12 pureanti-ASGPR mAbs. Cells were then tested for expression of cell surfaceCD86 (a DC activation marker) and supernatants were assayed for secretedcytokines. Three mAbs (36, 38, 43) from the anti-ASGPR mAb panelactivated DC.

FIG. 6 shows that different antigens can be expressed in the context ofa DC-ASGPR rAb. Such an anti-DC-ASGPR rAb.Doc protein can be simplymixed with any Cohesin.fusion protein to assemble a stable non-covalent[rAb.Doc:Coh.fusion] complex that functions just as a rAb.fusionprotein. FIG. 6 shows that such a [rAb.Doc:Coh.fusion] complex can focusantigen to the surface of cells expressing DC-ASGPR. The figure alsoshows anti-DC-ASGPR.Doc:Coh.Flu M1 complexes deliver Flu M1 to thesurface of 293F cells transfected with DC-ASGPR cDNA. 1 μg/ml ofanti-DC-ASGPR.Doc rAb (shown as the peak on the right) or controlhIgG4.Doc rAb (shown as the peak on the left) were incubated withbiotinylated Coh.Flu M1 (2 μg/ml) for 1 hr at R.T. transfected 293Fcells were added and incubation continued for 20 min on ice. Cells werethen washed and stained with PE-labeled streptavidin. Cells were thenanalyzed for PE fluorescence.

Anti-DC-ASGPR rAb complexed to Flu M1 via Dockerin:Cohesin interactiontargets the antigen to human DCs and results in the expansion of FluM1-specific CD8+ T cells—the potential utility of anti-DC-ASGPR rAbs asdevices to deliver antigen to e.g., DC is shown in the figure below.FIG. 7 shows the dramatic expansion of Flu M1-specific CD8+ cells ishighly predictive of potency of such an agent as a vaccine directed toeliciting protective immune responses against Flu M1.

FIGS. 8A-8D demonstrated the cross reactivity of the differentantibodies with monkey ASGPR. For pIRES_ASGPR-mon (monkey) was cloned byinserting the PCR product into NheI-NotI sites of pIRES vector. Thesequence of final product is base on clone 5S10. Most other clones areeither similar to this with one aa difference or identical to this.However, one clone, 5S1, has an A deletion near the 3′ end, whichgenerated a shortened and different C′ terminus and maybe used as asecond variant. To clone the monkey ASGPR, the following oligos wereused: DC-ASGPR_MoN:gaattcgctagcCACCATGACATATGAAAACTTCCAAGACTTGGAGAGTGAGGAGAAAGTC CAAGGGG(SEQ ID NO.: 15); and DC-ASGPR_Mo:CGAATTCGCGGCCGCTCAGTGACTCTCCTGGCTGGCCTGGGTCAGACCAGCCTCGCA GACCC (SEQ IDNO.: 16), which is a reverse complement ofGGGTCTGCGAGGCTGGTCTGACCCAGGCCAGCCAGGAGAGTCACTGAGCGGCCGCG AATTCG (SEQ IDNO.: 17). Sequence comparisons indicate the likely regions of overlapand, hence, the cross-reactivity, as is known to those if skill in theart.

The following table demonstrated the binding of the DC-ASGPR 334998 200ug/ml 12.05.07 cfg#558 anti-Human IgG PE

Avg StDev SEM w/o w/o w/o Glycan Max Max & Max & number Glycan name &Min Min Min % CV 82 GalNAcα1-3(Fucα1-2)Galβ1-4GlcNAcμ-Sp8 52930 102655132 19 210 Neu5Acα2-3(GalNAcβ1-4)Galβ1-4GlcNAcβ-Sp8 49937 4969 2484 1086 GalNAcα1-3Galβ-Sp8 49067 4672 2336 10 89 GalNAcβ1-3(Fucα1-2)Galβ-Sp847375 5453 2726 12 84 GalNAcα1-3(Fucα1-2)Galβ-Sp8 46555 6618 3309 14 209Neu5Acα2-3(GalNAcβ1-4)Galβ1-4GlcNAcβ-Sp0 46169 2121 1060 5 175GlcNAcβ1-6GalNAcα-Sp8 44809 1939 969 4 301 GalNAcα1-3(Fucα1-2)Galβ-Sp1844147 6003 3002 14 211 Neu5Acα2-3(GalNAcβ1-4)Galβ1-4Glcβ-Sp0 43603 35171759 8 10 α-GalNAc-Sp8 43514 2476 1238 6 128Galβ1-3GalNAcβ1-4(Neu5Acα2-3)Galβ1-4Glcβ-Sp0 43152 13339 6669 31 151Galβ1-4GlcNAcβ1-6GalNAcα-Sp8 42871 2466 1233 6 92 GalNAcβ1-4GlcNAcβ-Sp042845 3394 1697 8 93 GalNAcβ1-4GlcNAcβ-Sp8 41764 7340 3670 18 87GalNAcα1-4(Fucα1-2)Galβ1-4GlcNAcβ-Sp8 41584 2925 1462 7 79GalNAcα1-3(Fucα1-2)Galβ1-3GlcNAcβ-Sp0 41406 14134 7067 34 20β-GalNAc-Sp8 40803 2388 1194 6 206Neu5Acα2-8Neu5Acα2-3(GalNAcβ1-4)Galβ1-4Glcβ-Sp0 38720 2736 1368 7 242Neu5Acα2-6GalNAcα-Sp8 37500 1934 967 5 91 GalNAcβ1-4(Fucα1-3)GlcNAcβ-Sp037286 5046 2523 14 204 Neu5Acα2-8Neu5Acα2-8Neu5Acα2-3(GalNAcβ1-4)Galβ1-37237 995 497 3 4Glcβ-Sp0 203NeuAcα2-8NeuAcα2-8NeuAcα2-8NeuAcα2-3(GalNAcβ1- 36746 2399 1200 74)Galβ1-4Glcβ-Sp0 243 Neu5Acα2-6GalNAcβ1-4GlcNAcβ-Sp0 36375 1661 830 559 Fucα1-2Galβ1-3GalNAcβ1-4(Neu5Acα2-3)Galβ1-4Glcβ-Sp0 35701 6903 345219 90 GalNAcβ1-3Galα1-4Galβ1-4GlcNAcβ-Sp0 34350 760 380 2 83GalNAcα1-3(Fucα1-2)Galβ1-4Glcβ-Sp0 28846 9844 4922 34 302GalNAcβ1-3Galβ-Sp8 28745 15727 7864 55 300 GalNAcα-Sp15 18125 18847 9424104 127 Galβ1-3GalNAcβ1-3Galα1-4Galβ1-4Glcβ-Sp0 17999 9798 4899 54 85GalNAcα1-3GalNAcβ-Sp8 12643 10843 5422 86 173GlcNAcβ1-4GlcNAcβ1-4GlcNAcβ-Sp8 8673 940 470 11 81GalNAcα1-3(Fucα1-2)Galβ1-4GlcNAcβ-Sp0 7672 12937 6469 169 30[3OSO3]Galβ1-4(6OSO3)Glcβ-Sp8 7394 292 146 4 120Galβ1-3(Galβ1-4GlcNAcβ1-6)GalNAcα-Sp8 5664 1311 655 23 80GalNAcα1-3(Fucα1-2)Galβ1-4(Fucα1-3)GlcNAcβ-Sp0 5444 907 454 17 147Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ-Sp0 4927 410 205 8 29[3OSO3]Galβ1-4(6OSO3)Glcβ-Sp0 4871 908 454 19 101 Galα1-3GalNAcα-Sp84815 3163 1581 66 214 Neu5Acα2-3GalNAcα-Sp8 4109 569 284 14 287[3OSO3][4OSO3]Galβ1-4GlcNacβ-SpSp0 3959 1646 823 42 40[4OSO3]Galβ1-4GlcNAcβ-Sp8 3848 673 337 17 45[6OSO3]Galβ1-4[6OSO3]Glcβ-Sp8 3790 993 497 26 166GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ-SpO 3720 435 218 12 227Neu5Acα2-3Galβ1-4[6OSO3]GlcNAcβ-Sp8 3576 793 397 22 218NeuAcα2-3Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1-4(Fucα1- 3360 104 52 33)GlcNAcβSp0 240 Neu5Acα2-3Galβ1-4Glcβ-Sp8 3313 976 488 29 149Galβ1-4GlcNAcβ1-3Galβ1-4Glcβ-Sp8 3233 263 132 8 244Neu5Acα2-6Galβ1-4[6OSO3]GlcNAcβ-Sp8 3195 757 379 24 270Fucα1-2Galβ1-4[6OSO3]GlcNAc-Sp8 3161 2563 1282 81 42[6OSO3]Galβ1-4Glcβ-Sp0 3084 529 264 17 271Fucα1-2[6OSO3]Galβ1-4[6OSO3]Glc-Sp0 3063 377 188 12 172(GlcNAcβ1-4)5β-Sp8 3032 1058 529 35 47 [6OSO3]GlcNAcβ-Sp8 3008 159 80 5143 Neu5Acα2-3Galβ1-4GlcNAcβ1-2Manα13(Neu5Acα2- 3008 309 155 103Galβ1-4GlcNAcβ1-2Manα1-6)Manβ1-4GlcNAcβ1- 4GlcNAcβ-Sp12 265[3OSO3]Galβ1-4(Fucα1-3)(6OSO3)Glc-Sp0 2995 1841 921 61 139Galβ1-4[6OSO3]Glcβ-Sp0 2988 1070 535 36 27[3OSO3][6OSO3]Galβ1-4GlcNAcβ-Sp0 2930 317 158 11 273Fucα1-2-Galβ1-4[6OSO3]Glc-Sp0 2919 495 247 17 319Neu5Acα2-6Galβ1-4GlcNAcβ1-2Manα1-3(Galβ1- 2730 993 497 364GlcNAcβ1-2Manα1-6)Manβ1-4GlcNAcβ1-4GlcNAcβ-Sp12 35[3OSO3]Galβ1-4[6OSO3]GlcNAcβ-Sp8 2722 516 258 19 28[3OSO3]Galβ1-4Glcβ-Sp8 2674 197 98 7 38 [3OSO3]Galβ-Sp8 2652 1680 840 63253 Neu5Acα2-8Neu5Acα2-3Galβ1-4Glcβ-Sp0 2631 1136 568 43 2896-H2PO3Glcβ-Sp10 2611 674 337 26 26[3OSO3][6OSO3]Galβ1-4[6OSO3]GlcNAcβ-SpO 2550 153 76 6 266[3OSO3]Galβ1-4(Fucα1-3)Glc-Sp0 2529 444 222 18 54Neu5Acα2-6Galβ1-4GlcNAcβ1-2Manα1-3(Neu5Acα2- 2476 300 150 126Galβ1-4GlcNAcβ1-2Manα1-6)Manβ1-4GlcNAcβ1- 4GlcNAcβ-Sp8 303GlcAβ1-3GlcNAcβ-Sp8 2463 130 65 5 32 [3OSO3]Galβ1-3GalNAcα-Sp8 2461 622311 25 53 Neu5Acα2-6Galβ1-4GlcNAcβ1-2Manα1-3(Neu5Acα2- 2455 283 142 126Galβ1-4GlcNAcβ1-2Manα1-6)Manβ1-4GlcNAcβ1- 4GlcNAcβ-Sp13 181Glcβ1-6Glcβ-Sp8 2455 154 77 6 267[3OSO3]Galβ1-4[Fucα1-3][6OSO3]GlcNAc-Sp8 2447 1065 532 44 293Galβ1-3(Neu5Acα2-3Galβ1-4(Fucα1-3)GlcNAcβ1- 2359 648 324 276)GalNAc-Sp14 202 Neu5Acα2-3Galβ1-3GalNAcα-Sp8 2349 928 464 40 163GlcNAcβ1-3Galβ1-3GalNAcα-Sp8 2347 375 188 16 1 Neu5Acα2-8Neu5Acα-Sp82339 1539 769 66 31 [3OSO3]Galβ1-3(Fucα1-4)GlcNAcβ-Sp8 2332 319 160 14230 Neu5Acα2-3Galβ1-4(Fucα1-3)GlcNAcβ-Sp0 2306 164 82 7 286[3OSO3]Galβ1-4[6OSO3]GlcNAβ-Sp0 2290 472 236 21 318Neu5Acα2-3Galβ1-4GlcNAcβ1-2Manα1-3(Neu5Acα2- 2262 246 123 116Galβ1-4GlcNAcβ1-2Manα1-6)Manβ1-4GlcNAcβ1- 4GlcNAcβ-Sp12 199Neu5Acα2-6Galβ1-4GlcNAcβ1-2Manα1-3(Neu5Acα2- 2217 138 69 63Galβ1-4GlcNAcβ1-2Manα1-6)Manβ1-4GlcNAcβ1- 4GlcNAcβ-Sp12 39[4OSO3][6OSO3]Galβ1-4GlcNAcβ-Sp0 2215 619 310 28 77 Fucα1-4GlcNAcβ-Sp82207 83 42 4 285 Neu5Acα2-3Galβ1-4GlcNAcβ1-3Galβ1-3GlcNAcβ-Sp0 2193 1679839 77 262 Neu5Gcα2-6GalNAcα-Sp0 2192 734 367 33 216Neu5Acα2-3Galβ1-3(6OSO3)GlcNAc-Sp8 2163 1062 531 49 43[6OSO3]Galβ1-4Glcβ-Sp8 2149 700 350 33 297Galβ1-4GlcNAcβ1-3(GlcNAcβ1-6)Galβ1-4GlcNAc-Sp0 2141 983 491 46 224NeuAcα2-3Galβ1-3GlcNAcβ1-3Galβ1-4GlcNAcβ-Sp0 2133 1208 604 57 3Neu5Acα2-8Neu5Acα2-8Neu5Acβ-Sp8 2117 611 306 29 171 (GlcNAcβ1-4)6β-Sp82112 302 151 14 316 Neu5Acα2-3Galβ1-3(Neu5Acα2-6)GalNAc-Sp14 2105 1171585 56 15 α-Neu5Ac-Sp11 2099 250 125 12 52 Galβ1-4GlcNAcβ1-2Manα1-3(Galβ1-4GlcNAcβ1-2Manα1- 2092 429 215 21 6)Manβ1-4GlcNAcβ1-4GlcNAcβ-Sp13268 [3OSO3]Galβ1-4[Fucα1-3]GlcNAc-Sp0 2085 955 477 46 313Manα1-2Manα1-2Manα1-3(Manα1-2Manα1-6(Manα1- 2020 812 406 403)Manα1-6)Manα-Sp9 225 Neu5Acα2-3Galβ1-3GlcNAcβ-Sp0 2019 1052 526 52 36[3OSO3]Galβ1-4GlcNAcβ-Sp0 2012 389 194 19 263Neu5Gca2-6Galβ1-4GlcNAcβ-Sp0 1999 664 332 33 141Galβ1-4GalNAcαl-3(Fucα1-2)Galβ1-4GlcNAcβ-Sp8 1968 772 386 39 274Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1-3(Fucα1-4)GlcNAcβ- 1961 78 39 4 Sp0 275Galβ1-3-(Galβ1-4GlcNacβ1-6)GalNAc-Sp14 1953 409 205 21 7 α-D-Gal-Sp81925 636 318 33 41 6-H2PO3Manα-Sp8 1919 223 111 12 247Neu5Acα2-6Galβ1-4GlcNAcβ1-3Galβ1-4(Fucα1- 1914 169 85 93)GlcNAcβ1-3Galβ1-4(Fucα1-3)GlcNAcβ-Sp0 311 Manα1-6Manβ-Sp10 1906 522261 27 205 Neu5Acα2-8Neu5Acα2-8Neu5Acα2-3Galβ1-4Glcβ-Sp0 1902 222 111 12280 Galβ1-4[Fucα1-3][6OSO3]GlcNAc-Sp0 1881 982 491 52 152Galβ1-4GlcNAcβ-Sp0 1868 924 462 49 113 Galα1-6Glcβ-Sp8 1864 321 161 17115 Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1-4(Fucα1-3)GlcNAcβ- 1855 338 169 18Sp0 251 Neu5Acα2-6Galβ-Sp8 1842 316 158 17 116Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1-4GlcNAcβ-Sp0 1836 798 399 43 194Manα1-2Manα1-2Manα1-3(Manα1-2Manα1-3(Manα1- 1829 176 88 102Manα1-6)Manα1-6)Manβ1-4GlcNAcβ1-4GlcNAcβ-Sp12 33[3OSO3]Galβ1-3GlcNAcβ-Sp8 1812 889 445 49 272Fucα1-2-(6OSO3)-Galβ1-4Glc-Sp0 1805 86 43 5 207Neu5Acα2-8Neu5Acα2-8Neu5Acα-Sp8 1804 454 227 25 74 Fucα1-2Galβ-Sp8 1796648 324 36 213 Neu5Acα2-3(Neu5Acα2-6)GalNAcα-Sp8 1768 312 156 18 234Neu5Acα2-3Galβ1-4GlcNAcβ1-3Galβ1-4(Fucα1-3)GlcNAc- 1767 178 89 10 Sp0 50Manα1-3(Manα1-6)Manβ1-4GlcNAcβ1-4GlcNAcβ-Sp13 1759 553 277 31 111Galα1-4Galβ1-4Glcβ-Sp0 1740 635 318 36 291 Galα1-3GalNAcα-Sp16 1738 1090545 63 296 Galβ1-4GlcNAcβ1-3(Galβ1-4GlcNAcβ1-6)Galβ1-4GlcNAc- 1726 850425 49 Sp0 154 Galβ1-4Glcβ-Sp0 1725 457 229 27 56Fucα1-2Galβ1-3GalNAcβ1-3Galα1-4Galβ1-4Glcβ-Sp9 1719 384 192 22 66Fucα1-2Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4(Fucα1- 1703 224 112 133)GlcNAcβ1-3Galβ1-4(Fucα1-3)GlcNAcβ-Sp0 299Galβ1-4GlcNAcβ1-6Galβ1-4GlcNAcβ-Sp0 1658 820 410 49 44[6OSO3]Galβ1-4GlcNAcβ-Sp8 1632 242 121 15 237Neu5Acα2-3Galβ1-4GlcNAcβ-Sp8 1632 1049 524 64 233Neu5Acα2-3Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1- 1620 862 431 53 4GlcNAcβ-Sp8192 Manα1-6(Manα1-2Manα1-3)Manα1-6(Manα2Manα1- 1608 903 452 563)Manβ1-4GlcNAcβ1-4GlcNAcβ-Sp12 64 Fucα1-2Galβ1-3GlcNAcβ-Sp8 1602 625313 39 62 Fucα1-2Galβ1-3GlcNAcβ1-3Galβ1-4Glcβ-Sp8 1580 417 208 26 148Galβ1-4GlcNAcβ1-3Galβ1-4Glcβ-Sp0 1568 617 308 39 295Galβ1-4GlcNAcβ1-2Manα1-3(Neu5Acα2-6Galβ1- 1556 190 95 124GlcNAcβ1-2Manα1-6)Manβ1-4GlcNAcβ1-4GlcNAcβ-Sp12 137Galβ1-4(Fucα1-3)GlcNAcβ1-4Galβ1-4(Fucα1-3)GlcNAcβ- 1552 1313 656 85 Sp017 β-D-Gal-Sp8 1544 871 435 56 168 GlcNAcβ1-4MDPLys 1542 345 172 22 254Neu5Acβ2-6GalNAcα-Sp8 1541 688 344 45 231Neu5Acα2-3Galβ1-4(Fucα1-3)GlcNAcβ-Sp8 1534 257 129 17 125Galβ1-3GalNAcα-Sp8 1483 1025 512 69 269 Fucα1-2[6OSO3]Galβ1-4GlcNAc-Sp01473 191 96 13 182 G-ol-Sp8 1471 264 132 18 37 [3OSO3]Galβ1-4GlcNAcβ-Sp81462 1187 593 81 229 Neu5Acα2-3Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4(Fucα1 -1451 333 167 23 3)GlcNAcβ1-3Galβ1-4(Fucα1-3)GlcNAcβ-Sp0 315Neu5Acα2-3Galβ1-3(Neu5Acα2-3Galβ1-4GlcNAcβ1- 1448 1476 738 1026)GalNAc-Sp14 65 Fucα1-2Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4(Fucα1- 1442748 374 52 3)GlcNAcβ-Sp0 164 GlcNAcβ1-3Galβ1-4GlcNAcβ-Sp0 1436 1332 66693 305 GlcNAcβ1-2Manα1-3(GlcNAcβ1-2Manα1-6)Manβ1- 1428 288 144 204GlcNAcβ1-4GlcNAcβ-Sp12 304 GlcNAcβ1-2Manα1-3(Neu5Acα2-6Galβ1-4GlcNAcβ1-1428 499 249 35 2Manα1-6)Manβ1-4GlcNAcβ1-4GlcNAcβ-Sp12 145Galβ1-4GlcNAcβ1-3Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1- 1422 323 162 234(Fucα1-3)GlcNAcβ-Sp0 117 Galβ1-3(Fucα1-4)GlcNAc-Sp0 1407 681 341 48 193Manα1-2Manα1-6(Manα1-3)Manα1-6(Manα2Manα2Manα1- 1404 285 142 203)Manβ1-4GlcNAcβ1-4GlcNAcβ-Sp12 19 β-D-Man-Sp8 1389 635 317 46 176GlcNAcβ1-6Galβ1-4GlcNAcβ-Sp8 1383 1000 500 72 232Neu5Acα2-3Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ-Sp8 1355 374 187 28 219Neu5Acα2-3Galβ1-3(Neu5Acα2-3Galβ1-4)GlcNAcβ-Sp8 1350 753 377 56 123Galβ1-3(Neu5Acβ2-6)GalNAcα-Sp8 1350 852 426 63 276Galβ1-3(GlcNacβ1-6)GalNAc-Sp14 1345 353 176 26 208Neu5Acα2-3(6-O-Su)Galβ1-4(Fucα1-3)GlcNAcβ-Sp8 1341 642 321 48 55Fucα1-2Galβ1-3GalNAcβ1-3Galα-Sp9 1331 466 233 35 257Neu5Gca2-3Galβ1-3(Fucα1-4)GlcNAcβ-Sp0 1315 108 54 8 201Fucα1-3(Galβ1-4)GlcNAcβ1-2Manα1-3(Fucα1-3(Galβ1- 1294 289 144 224)GlcNAcβ1-2Manα1-6)Manβ1-4GlcNAcβ1-4GlcNAcβ- Sp20 97Galα1-3(Fucα1-2)Galβ1-4GlcNAc-Sp0 1282 583 291 45 150Galβ1-4GlcNAcβ1-6(Galβ1-3)GalNAcα-Sp8 1265 778 389 62 60Fucα1-2Galβ1-3GalNAcβ1-4(Neu5Acα2-3)Galβ1-4Glcβ-Sp9 1261 738 369 59 317Neu5Acα2-3Galβ1-3GalNAc-Sp14 1239 780 390 63 23 β-GlcN(Gc)-Sp8 1219 436218 36 279 Galβ1-3GlcNAcβ1-3Galβ1-3GlcNAcβ-Sp0 1219 570 285 47 190Manα1-2Manα1-3(Manα1-2Manα1-6)Manα-Sp9 1217 1305 653 107 178Glcα1-4Glca-Sp8 1216 560 280 46 146Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ- 1211 1315 658 109 Sp0292 Galβ1-3GalNAcα-Sp16 1198 370 185 31 221Neu5Acα2-3Galβ1-3(Neu5Acα2-6)GalNAcα-Sp8 1194 238 119 20 99Galα1-3(Fucα1-2)Galβ-Sp8 1189 767 383 64 309HOOC(CH3)CH-3-O-GlcNAcβ1-4GlcNAcβ-Sp10 1186 1108 554 93 248Neu5Acα2-6Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ-Sp0 1181 334 167 28 107Galα1-3Galβ-Sp8 1148 688 344 60 236 Neu5Acα2-3Galβ1-4GlcNAcβ-Sp0 1148441 220 38 320 Neu5Acα2-6Galβ1-4GlcNAcβ1-2Manα1-3(GlcNAcβ1- 1142 55 27 52Manα1-6)Manβ1-4GlcNAcβ1-4GlcNAcβ-Sp12 197Manα1-6(Manα1-3)Manα1-6(Manα2Manα1-3)Manβ1- 1134 200 100 184GlcNAcβ1-4GlcNAcβ-Sp12 185 GlcAβ1-3Galβ-Sp8 1133 470 235 42 34[3OSO3]Galβ1-4(Fucα1-3)GlcNAcβ-Sp8 1117 980 490 88 109Galα1-4Galβ1-4GlcNAcβ-Sp0 1094 499 250 46 235Neu5Acα2-3Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1- 1092 1077 539 994GlcNAcβ-Sp0 228 Neu5Acα2-3Galβ1-4(Fucα1-3)(6OSO3)GlcNAcβ-Sp8 1090 771385 71 184 GlcAβ-Sp8 1072 476 238 44 282Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-3(Fucα1-4)GlcNAcβ- 1062 239 120 23 Sp0 2Neu5Acα2-8Neu5Acβ-Sp17 1060 84 42 8 174 GlcNAcβ1-6(Galβ1-3)GalNAcα-Sp81039 913 456 88 261 Neu5Gcα2-3Galβ1-4Glcβ-Sp0 1034 440 220 43 18β-D-Glc-Sp8 1024 335 167 33 217 Neu5Acα2-3Galβ1-3(Fucα1-4)GlcNAcβ-Sp81023 646 323 63 260 Neu5Gcα2-3Galβ1-4GlcNAcβ-Sp0 1020 208 104 20 104Galα1-3Galβ1-3GlcNAcβ-Sp0 1017 297 149 29 245Neu5Acα2-6Galβ1-4GlcNAcβ-Sp0 1010 394 197 39 14 α-Neu5Ac-Sp8 998 1046523 105 283 Galβ1-4GlcNAcβ1-3Galβ1-3GlcNAcβ-Sp0 978 514 257 53 156GlcNAcαl -3Galβ1-4GlcNAcβ-Sp8 969 276 138 29 310Manα1-3(Manα1-6)Manβ1-4GlcNAcβ1-4GlcNAcβ-Sp12 965 238 119 25 183GlcAa-Sp8 960 463 232 48 138 Galβ1-4(Fucα1-3)GlcNAcβ1-4Galβ1-4(Fucα1-3)GlcNAcβ1- 948 595 297 634Galβ1-4(Fucα1-3)GlcNAcβ-Sp0 96Galα1-3(Fucα1-2)Galβ1-4(Fucα1-3)GlcNAcβ-Sp0 948 260 130 27 6Neu5Acα2-6Galβ1-4GlcNAcβ1-2Manα1-3(Neu5Acα2- 943 351 176 376Galβ1-4GlcNAcβ1-2Manα1-6)Manβ1-4GlcNAcβ1- 4GlcNAcβ-Sp12 306GlcNAcβ1-3Man-Sp10 938 153 77 16 121 Galβ1-3(GlcNAcβ1-6)GalNAcα-Sp8 936748 374 80 258 Neu5Gca2-3Galβ1-3GlcNAcβ-Sp0 932 375 188 40 246Neu5Acα2-6Galβ1-4GlcNAcβ-Sp8 931 635 317 68 200 Manβ1-4GlcNAcβ-Sp0 920322 161 35 78 Fucβ1-3GlcNAcβ-Sp8 911 464 232 51 94 Galα1-2Galβ-Sp8 911393 197 43 256 Galβ1-4GlcNAcβ1-2Manα1-3 (Neu5Acα2-6Galβ1- 909 428 214 474GlcNAcβ1-2Manα1-6)Manβ1-4GlcNAcβ1-4GlcNAcβ-Sp21 95Galα1-3(Fucα1-2)Galβ1-3GlcNAcβ-Sp0 908 245 123 27 8 α-D-Glc-Sp8 904 417209 46 103 Galα1-3Galβ1-4(Fucα1-3)GlcNAcβ-Sp8 893 445 222 50 118Galβ1-3(Fucα1-4)GlcNAc-Sp8 890 624 312 70 9 α-D-Man-Sp8 881 403 201 4616 β-Neu5Ac-Sp8 876 935 468 107 119 Galβ1-3(Fucα1-4)GlcNAcβ-Sp8 872 283141 32 278 Galβ1-3GalNAc-Sp14 851 144 72 17 187KDNα2-3Galβ1-3GlcNAcβ-Sp0 839 386 193 46 69Fucα1-2Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAc-Sp0 837 328 164 39 76Fucα1-3GlcNAcβ-Sp8 836 276 138 33 108 Galα1-4(Fucα1-2)Galβ1-4GlcNAcβ-Sp8819 58 29 7 212 NeuAcα2-3(NeuAcα2-3Galβ1-3GalNAcβ1-4)Galβ1-4Glcβ- 8181442 721 176 Sp0 132 Galβ1-3GlcNAcβ1-3Galβ1-4Glcβ-Sp10 816 353 176 43105 Galα1-3Galβ1-4GlcNAcβ-Sp8 806 184 92 23 308 GlcNAcβ1-4GlcNAcβ-Sp12796 360 180 45 160 GlcNAcβ1-3(GlcNAcβ1-6)Galβ1-4GlcNAcβ-Sp8 794 416 20852 284 Neu5Acα2-3Galβ1-3GlcNAcβ1-3Galβ1-3GlcNAcβ-Sp0 777 491 245 63 188KDNα2-3Galβ1-4GlcNAcβ-Sp0 774 320 160 41 215Neu5Acα2-3GalNAcβ1-4GlcNAcβ-Sp0 762 252 126 33 294Galβ1-3Galβ1-4GlcNAcβ-Sp8 746 255 128 34 196Manα1-3(Manα1-2Manα1-2Manα1-6)Manα-Sp9 744 177 88 24 189Manα1-2Manα1-2Manα1-3Manα-Sp9 743 207 103 28 25GlcNAcβ1-3(GlcNAcβ1-4)(GlcNAcβ1-6)GlcNAc-Sp8 735 270 135 37 131Galβ1-3GlcNAcβ1-3Galβ1-4GlcNAcβ-Sp0 728 290 145 40 277Galβ1-3-(Neu5Aa2-3Galβ1-4GlcNacβ1-6)GalNAc-Sp14 722 324 162 45 136Galβ1-4(Fucα1-3)GlcNAcβ-Sp8 718 93 46 13 70Fucα1-2Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1- 713 861 430 1214GlcNAcβ-Sp0 110 Galα1-4Galβ1-4GlcNAcβ-Sp8 712 183 91 26 129Galβ1-3GalNAcβ1-4Galβ1-4Glcβ-Sp8 702 224 112 32 71Fucα1-2Galβ1-4GlcNAcβ-Sp0 686 160 80 23 169GlcNAcβ1-4(GlcNAcβ1-6)GalNAcα-Sp8 686 229 115 33 122Galβ1-3(Neu5Acα2-6)GalNAcα-Sp8 679 157 79 23 106 Galα1-3Galβ1-4Glcβ-Sp0678 137 69 20 255 Neu5Acβ2-6Galβ1-4GlcNAcβ-Sp8 671 153 76 23 130Galβ1-3Galβ-Sp8 668 285 143 43 144 Galβ1-4GlcNAcβ1-3GalNAcα-Sp8 663 227113 34 13 α-L-Rhα-Sp8 662 245 123 37 22 β-GlcNAc-Sp8 655 313 157 48 72Fucα1-2Galβ1-4GlcNAcβ-Sp8 646 95 47 15 157 GlcNAcα1-6Galβ1-4GlcNAcβ-Sp8644 323 162 50 307 GlcNAcβ1-4GlcNAcβ-Sp10 640 336 168 53 180Glcβ1-4Glcβ-Sp8 608 316 158 52 191 Manα1-2Manα1-3Manα-Sp9 607 104 52 17134 Galβ1-3GlcNAcβ-Sp8 603 103 51 17 21 β-GlcNAc-Sp0 595 285 142 48 24(Galβ1-4GlcNAcβ)2-3,6-GalNAcα-Sp8 590 240 120 41 223NeuAcα2-3Galβ1-3GalNAcβ1-3Galα1-4Galβ1-4Glcβ-Sp0 580 191 95 33 162GlcNAcβ1-3Galβ-Sp8 577 435 217 75 135 Galβ1-4(Fucα1-3)GlcNAcβ-Sp0 561139 70 25 249 Neu5Acα2-6Galβ1-4Glcβ-Sp0 560 377 189 67 489NAcNeu5Acα-Sp8 556 470 235 85 158 GlcNAcβ1-2Galβ1-3GalNAcα-Sp8 550 417208 76 264 Neu5Gcα-Sp8 550 305 152 55 46NeuAcα2-3[6OSO3]Galβ1-4GlcNAcβ-Sp8 545 363 182 67 68Fucα1-2Galβ1-4(Fucα1-3)GlcNAcβ-Sp8 541 208 104 38 222 Neu5Acα2-3Galβ-Sp8526 277 139 53 298 Galβ1-4GlcNAcαl -6Galβ1-4GlcNAcβ-Sp0 494 335 167 6898 Galα1-3(Fucα1-2)Galβ1-4Glcβ-Sp0 482 112 56 23 312Manα1-6(Manα1-3)Manα1-6(Manα1-3)Manβ-Sp10 453 292 146 64 133Galβ1-3GlcNAcβ-Sp0 452 165 82 36 57 Fucα1-2Galβ1-3(Fucα1-4)GlcNAcβ-Sp8450 268 134 60 114 Galβ1-2Galβ-Sp8 449 324 162 72 198Manα1-6(Manα1-3)Manα1-6(Manα1-3)Manβ1-4GlcNAcβ1-4 448 204 102 45GlcNAcβ-Sp12 161 GlcNAcβ1-3GalNAcα-Sp8 442 156 78 35 281Galβ1-4[Fucα1-3][6OSO3]Glc-Sp0 439 144 72 33 259Neu5Gca2-3Galβ1-4(Fucα1-3)GlcNAcβ-Sp0 433 357 179 83 67Fucα1-2Galβ1-4(Fucα1-3)GlcNAcβ-Sp0 420 94 47 22 12 α-L-Fuc-Sp9 410 303151 74 159 GlcNAcβ1-3(GlcNAcβ1-6)GalNAcα-Sp8 407 88 44 22 75Fucα1-3GlcNAcβ-Sp8 399 182 91 46 239 Neu5Acα2-3Galβ1-4Glcβ-Sp0 395 15678 39 290 Galα1-3(Fucα1-2)Galβ-Sp18 389 246 123 63 11 α-L-Fuc-Sp8 387231 115 60 51 GlcNAcβ1-2Manα1-3(GlcNAcβ1-2Manα1-6)Manβ1- 383 164 82 434GlcNAcβ1-4GlcNAcβ-Sp13 5Galβ1-3GlcNAcβ1-2Manα1-3(Galβ1-3GlcNAcβ1-2Manα1- 381 529 265 1396)Manβ1-4GlcNAcβ1-4GlcNAcβ-Sp19 63 Fucα1-2Galβ1-3GlcNAcβ-Sp0 362 187 9352 241 Galβ1-4GlcNAcβ1-2Manα1-3(Fucα1-3(Galβ1-4)GlcNAcβ1- 352 68 34 192Manα1-6)Manβ1-4GlcNAcβ1-4GlcNAcβ-Sp20 155 Galβ1-4Glcβ-Sp8 315 105 53 33126 Galβ1-3GalNAcβ-Sp8 288 265 132 92 195 Manα1-3(Manα1-6)Manα-Sp9 26992 46 34 88 GalNAcβ1-3GalNAcα-Sp8 262 107 54 41 252Neu5Acα2-8Neu5Acα-Sp8 260 214 107 82 167 GlcNAcβ1-3Galβ1-4Glcβ-Sp0 257129 64 50 140 Galβ1-4Ξ6OSO3]Glcβ-Sp8 256 345 172 135 177 Glcα1-4Glcβ-Sp8246 113 57 46 179 Glcα1-6Glcα1-6Glcβ-Sp8 225 380 190 168 314Manα1-2Manα1-2Manα1-3(Manα1-2Manα1-6(Manα1- 221 329 165 1492Manα1-3)Manα1-6)Manα-Sp9 238Neu5Acα2-3Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ-Sp0 212 200 100 94 220Neu5Acα2-3Galβ1-3[6OSO3]GalNAcα-Sp8 210 153 77 73 142Galβ1-4GalNAcβ1-3(Fucα1-2)Galβ1-4GlcNAcβ-Sp8 204 126 63 62 61Fucα1-2Galβ1-3GlcNAcβ1-3Galβ1-4Glcβ-Sp10 196 67 34 34 102Galα1-3GalNAcβ-Sp8 188 198 99 105 170 GlcNAcβ1-4Galβ1-4GlcNAcβ-Sp8 184127 64 69 124 Galβ1-3(Neu5Acα2-6)GlcNAcβ1-4Galβ1-4Glcβ-Sp10 173 146 7384 100 Galα1-3(Galα1-4)Galβ1-4GlcNAcβ-Sp8 168 112 56 66 186GlcAβ1-6Galβ-Sp8 158 171 86 108 4 Neu5Gcβ2-6Galβ1-4GlcNAc-Sp8 152 96 4863 73 Fucα1-2Galβ1-4Glcβ-Sp0 148 205 103 139 499NAcNeu5Acα2-6Galβ1-4GlcNAcβ-Sp8 146 159 79 108 58Fucα1-2Galβ1-3GalNAcα-Sp8 136 171 86 126 250 Neu5Acα2-6Galβ1-4Glcβ-Sp8122 144 72 119 112 Galα1-4GlcNAcβ-Sp8 115 82 41 72 165GlcNAcβ1-3Galβ1-4GlcNAcβ-Sp8 84 68 34 81 226Neu5Acα2-3Galβ1-3GlcNAcβ-Sp8 76 85 42 112 288[6OSO3]Galβ1-4[6OSO3]GlcNacβ-Sp0 72 130 65 180 153 Galβ1-4GlcNAcβ-Sp8 4858 29 120

It is contemplated that any embodiment discussed in this specificationcan be implemented with respect to any method, kit, reagent, orcomposition of the invention, and vice versa. Furthermore, compositionsof the invention can be used to achieve methods of the invention.

It will be understood that particular embodiments described herein areshown by way of illustration and not as limitations of the invention.The principal features of this invention can be employed in variousembodiments without departing from the scope of the invention. Thoseskilled in the art will recognize, or be able to ascertain using no morethan routine experimentation, numerous equivalents to the specificprocedures described herein. Such equivalents are considered to bewithin the scope of this invention and are covered by the claims.

All publications and patent applications mentioned in the specificationare indicative of the level of skill of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.” The use of the term “or” in the claims isused to mean “and/or” unless explicitly indicated to refer toalternatives only or the alternatives are mutually exclusive, althoughthe disclosure supports a definition that refers to only alternativesand “and/or.” Throughout this application, the term “about” is used toindicate that a value includes the inherent variation of error for thedevice, the method being employed to determine the value, or thevariation that exists among the study subjects.

As used in this specification and claim(s), the words “comprising” (andany form of comprising, such as “comprise” and “comprises”), “having”(and any form of having, such as “have” and “has”), “including” (and anyform of including, such as “includes” and “include”) or “containing”(and any form of containing, such as “contains” and “contain”) areinclusive or open-ended and do not exclude additional, unrecitedelements or method steps.

The term “or combinations thereof” as used herein refers to allpermutations and combinations of the listed items preceding the term.For example, “A, B, C, or combinations thereof” is intended to includeat least one of: A, B, C, AB, AC, BC, or ABC, and if order is importantin a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB.Continuing with this example, expressly included are combinations thatcontain repeats of one or more item or term, such as BB, AAA, MB, BBC,AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan willunderstand that typically there is no limit on the number of items orterms in any combination, unless otherwise apparent from the context.

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

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1.-30. (canceled)
 31. A method of delivering an antigen to an antigenpresenting cell, comprising administering to the antigen presenting cellan effective amount of a composition comprising a recombinant fusionprotein, said fusion protein comprising an anti-human dendritic cellasialoglycoprotein receptor (ASGPR) antibody or an antigen bindingfragment thereof and one or more autoimmune antigens, wherein theantibody or fragment thereof comprises (a) an immunoglobulin heavy chainselected from the group consisting of SEQ ID NO: 5, 7, 9, and 11; and(b) an immunoglobulin light chain selected from the group consisting ofSEQ ID NO: 6, 8, 10, and
 12. 32. The method of claim 31, wherein theantigen is Smith protein, RNP ribonucleoprotein, SS-A protein, SS-Bprotein, acetylcholine receptor, luteinizing hormone, follicularstimulating hormone, testosterone, growth hormone, prolactin, glutamicacid decarboxylase 65 (GAD 65), myelin basic protein, myelin proteolipidprotein, thyroglobulin, or thyroid stimulating hormone (TSH) receptor.33. The method of claim 32, wherein the antigen is myelin basic protein.34. The method of claim 31, wherein the antigen presenting cell is adendritic cell.
 35. The method of claim 31, wherein the antibody orfragment thereof comprises SEQ ID NO: 7 and SEQ ID NO:
 8. 36. The methodof claim 31, wherein the antibody or fragment thereof comprises SEQ IDNO: 9 and SEQ ID NO:
 10. 37. The method of claim 31, wherein theantibody or fragment thereof comprises SEQ ID NO: 11 and SEQ ID NO: 12.